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EP2263691A1 - Traitement de cancer avec l'anti-erbb2 anticorps recombinant humanisé monoclonal (rhuMAb 2C4) - Google Patents

Traitement de cancer avec l'anti-erbb2 anticorps recombinant humanisé monoclonal (rhuMAb 2C4) Download PDF

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Publication number
EP2263691A1
EP2263691A1 EP10176072A EP10176072A EP2263691A1 EP 2263691 A1 EP2263691 A1 EP 2263691A1 EP 10176072 A EP10176072 A EP 10176072A EP 10176072 A EP10176072 A EP 10176072A EP 2263691 A1 EP2263691 A1 EP 2263691A1
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Prior art keywords
antibody
her2
erbb2
antibodies
cells
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EP2263691B1 (fr
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Hans Koll
Birgit Bossenmaier
Hans-Joachim Müller
X. Sliwkowski Mark
Stephen Michael Kelsey
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
Genentech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G01N33/5759—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/485—Epidermal growth factor [EGF] (urogastrone)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to methods of identifying a tumor as responsive to treatment with anti-HER2 antibodies, as well as methods of treating patients suffering from such tumors.
  • the ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival.
  • the receptor family includes four distinct members including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185 neu ), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
  • EGFR epidermal growth factor receptor
  • HER2 ErbB2 or p185 neu
  • HER3 ErbB3
  • HER4 ErbB4 or tyro2
  • EGFR encoded by the erbB1 gene
  • EGFR has been causally implicated in human malignancy.
  • increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas.
  • Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF- ⁇ ), by the same tumor cells resulting in receptor activation by an autocrine stimulatory pathway.
  • TGF- ⁇ transforming growth factor alpha
  • ERRP epidermal growth factor receptor related protein
  • TGF- ⁇ and EGF Monoclonal antibodies directed against the EGFR or its ligands, TGF- ⁇ and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g ., Baselga and Mendelsohn., supra; Masui et al., Cancer Research, 44:1002-1007 (1984 ); and Wu et al., J. Clin. Invest., 95:1897-1905 (1995 ).
  • the second member of the ErbB family, p185neu was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats.
  • the activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein.
  • Amplification of the human homolog of neu (HER2) is observed in breast and ovarian cancers and correlates with a poor prognosis ( Slamon et al., Science, 235:177-182 (1987 ); Slamon et al., Science, 244:707-712 (1989 ); and US Patent No. 4,968,603 ).
  • ErbB2 may be overexpressed in prostate cancer ( Gu et al., Cancer Lett., 99:185-9 (1996 ); Ross et al., Hum. Pathol., 28:827-33 (1997 ); Ross et al., Cancer, 79:2162-70 (1997 ); and Sadasivan et al., J. Urol., 150:126-31 (1993 )). Overexpression of ErbB2 may lead to tumor growth via ligand-independent activation of ErbB2 or ErbB2 homodimers.
  • Hudziak et al., Mol. Cell. Biol., 9(3):1165-1172 (1989 ) describe the generation of a panel of anti-ErbB2 antibodies which were characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation of the SK-BR-3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize ErbB2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF- ⁇ . See also U.S. Patent No.
  • a recombinant humanized version of the murine anti-ErbB2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2 or HERCEPTIN ® ; U.S. Patent No. 5,821,337 ) is clinically active in patients with ErbB2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy ( Baselga et al., J. Clin. Oncol., 14:737-744 (1996 )).
  • HERCEPTIN ® received marketing approval from the Food and Drug Administration September 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the ErbB2 protein. However, not all ErbB2 overexpressing tumors respond to HERCEPTIN ® .
  • HERCEPTIN ® may be therapeutically effective in treating non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • HER2 protein is overexpressed in 20-66% of resected NSCLC tumors and has been shown to predict poor patient outcome in multiple series ( Azzoli, C.G. et al., Semin. Oncol., 29(Suppl 4):59-65 (2002 )).
  • ErbB3 U.S. Patent Nos. 5,183,884 and 5,480,968 as well as Kraus et al., PNAS (USA), 86:9193-9197 (1989 )
  • ErbB4 EP Pat Appln No 599,274 ; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993 ); and Plowman et al., Nature, 366:473-475 (1993 )). Both of these receptors display increased expression on at least some breast cancer cell lines.
  • the ErbB receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of ErbB ligands ( Earp et al., Breast Cancer Research and Treatment, 35:115-132 (1995 )). However, the mechanism by which these receptors aggregate and how this contributes to signaling is not fully understood ( Brennan, P.J. et al., Oncogene, 19:6093-6101 (2000 )).
  • EGFR is bound by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF- ⁇ ), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin ( Groenen et al., Growth Factors, 11:235-257 (1994 )).
  • EGF epidermal growth factor
  • TGF- ⁇ transforming growth factor alpha
  • HB-EGF heparin binding epidermal growth factor
  • betacellulin betacellulin and epiregulin
  • a family of heregulin proteins resulting from alternative splicing of a single gene are ligands for ErbB3 and ErbB4.
  • the heregulin family includes alpha, beta and gamma heregulins ( Holmes et al., Science, 256:1205-1210 (1992 ); U.S. Patent No.
  • neuregulin-2 (NRG-2) which is reported to bind either ErbB3 or ErbB4 ( Chang et al., Nature, 387:509-512 (1997 ); and Carraway et al., Nature, 387:512-516 (1997 )
  • neuregulin-3 which binds ErbB4
  • neuregulin-4 which binds ErbB4 ( Harari et al., Oncogene, 18:2681-89 (1999 )) HB-EGF, betacellulin and epiregulin also bind to ErbB4.
  • EGF and TGF ⁇ do not bind ErbB2, EGF stimulates EGFR and ErbB2 to form a heterodimer, which activates EGFR and results in transphosphorylation of ErbB2 in the heterodimer. Dimerization and/or transphosphorylation appears to activate the ErbB2 tyrosine kinase. See Earp et al., supra.
  • heregulin does not bind ErbB2, but when ErbB3 is co-expressed with ErbB2, an active signaling complex is formed ( Nagy et al., Cytometry, 32:120-31 (1998 ). Antibodies directed against ErbB2 are capable of disrupting this complex ( Sliwkowski et al., J.
  • ErbB3 is tyrosine kinase defective and thus requires heterodimerization, preferably with ErbB2, for signal transduction capabilities.
  • HRG heregulin
  • ErbB2 is the preferred heterodimerization partner for both EGFR and ErbB3.
  • ErbB4 like ErbB3, forms an active signaling complex with ErbB2 ( Carraway and Cantley, Cell, 78:5-8 (1994 )).
  • Ligand-dependent heterodimerization of ErbB2 with EGFR or ErbB3 may promote the growth of tumors that express ErbB2.
  • the present invention relates to a method of identifying a tumor that is responsive to treatment with an anti-HER2 antibody.
  • the anti-HER2 antibody blocks ligand activation of an ErbB heterodimer comprising HER2.
  • the antibody is monoclonal antibody 2C4, more preferably rhuMAb 2C4.
  • a sample of the tumor is obtained and the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complex is detected in the sample.
  • a tumor is identified as responsive to treatment with the anti-HER2 antibody when a complex is detected.
  • the presence of a complex is detected by immunoprecipitating any protein complexes that comprise HER2 with an anti-HER2 antibody.
  • the immunoprecipitated complexes are then contacted with an antibody selected from the group consisting of anti-HER3 antibodies, anti-HER1 antibodies, and anti-HER4 antibodies, and any binding is determined.
  • a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 complex is detected if it is determined that anti-HER3 and/or anti-HER1 and/or anti-HER4 antibodies bind to the immunoprecipitated complexes.
  • the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complex is detected by contacting the tumor sample with an anti-HER2 antibody that comprises a first fluorophore.
  • the tumor sample is then contacted with an antibody selected from the group consisting of anti-HER3 and/or anti-HER1 and/or anti-HER4 antibodies, wherein said antibody comprises a second fluorophore.
  • Measurements of fluorescence resonance energy transfer are then made to determine if the first fluorophore and the second fluorophore are in close proximity.
  • the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complex is detected if the first and second fluorophore are determined to be in close proximity.
  • the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 complex is detected by contacting the tumor sample with a first binding compound.
  • the first binding compound comprises a first target binding moiety that specifically binds HER2.
  • the first target binding moiety is preferably an anti-HER2 antibody or antibody fragment.
  • the first binding compound further comprises a detectable moiety that is linked to the first binding domain by a cleavable linker.
  • the tumor sample is contacted with a second binding compound.
  • the second binding compound preferably comprises a second target binding moiety that specifically binds HER3 or HER1 or HER4 and preferably does not bind HER2.
  • the second binding compound binds HER3 or HER1, and does not bind HER2 or HER4.
  • the second target binding moiety comprises an anti-HER3 or anti-HER1 or anti-HER4 antibody or antibody fragment.
  • the second binding compound is capable of cleaving the cleavable linker in the first binding compound upon activation, thus producing free detectable moiety if the first binding compound and the second binding compound are in close proximity.
  • the presence of a HER2/HER3 or HER2/HER1 or HER2/HER4 protein complex is detected when the presence of the free detectable moiety is identified.
  • the presence of the free detectable moiety is identified by capillary electrophoresis.
  • the first binding compound comprises a first target binding domain that specifically binds HER1 or HER3 or HER4, and the second binding compound comprises a second target binding domain that specifically binds HER2.
  • the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complex, and resultant HER2 activation is detected by assessing phosphorylation of ErbB receptor, for example by immunoprecipitation of the HER2 protein followed by Western blot immunodetection of phosphotyrosine.
  • the tumor sample that is analyzed for the presence of HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complexes is preferably obtained from a patient that is suffering from the tumor.
  • the sample can, for example, be obtained by biopsy.
  • the sample is obtained by purifying circulating tumor cells from the patient.
  • the sample is obtained during surgery to remove the tumor from the patient.
  • the sample of the tumor is obtained from a mammal other than the patient that originally developed the tumor.
  • the sample is obtained from a mouse, or another rodent.
  • the tumor is a xenografted tumor.
  • the xenografted tumor is preferably produced by transplanting a fragment of a human tumor into a mouse, or another rodent.
  • the tumor is a lung tumor, more preferably a non-small cell lung cancer tumor. In another embodiment, the tumor is a mammary tumor.
  • the invention concerns a method for identifying tumor cells as responsive to treatment with an antibody inhibiting the association of HER2 with another member of the ErbB receptor family, comprising the steps of (a) providing a biological sample comprising HER2-positive tumor cells; and (b) detecting the phosphorylation of an ErbB receptor in the biological sample, wherein said phosphorylation indicates that the tumor cells are responsive to treatment with the antibody.
  • the phosphorylation of an ErbB2 (HER2) receptor is detected.
  • the other member associated with HER2 is HER3, HER1, and/or HER4, such as HER2 and/or HER1.
  • the method can additionally comprise a step of detecting the presence of a HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complex, essentially as described above.
  • the invention further concerns a method for predicting the response of a subject diagnosed with a HER2-positive tumor to treatment with an antibody inhibiting the association of HER2 with another member of the ErbB receptor family, by detecting the formation of HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 protein complexes and/or the phosphorylation of an ErbB receptor in a biological sample obtained from the subject, comprising HER2-positive tumor cells.
  • the presence of such protein complexes and/or said phosphorylation indicates that the subject is likely to respond to treatment with the antibody.
  • the detection of the phosphorylation of ErbB2 (HER2) receptor indicates that the subject is likely to respond to treatment with the antibody.
  • the invention concerns a method for identifying a subject responsive to treatment with an anti-HER2 antibody, by detecting phosphorylation of an ErbB receptor in circulating tumor cells of the subject. The presence of such phosphorylation indicates that the subject is likely to respond to treatment with an anti-HER2 antibody.
  • the ErbB2 (HER2) phosphorylation is detected.
  • the subject is a human.
  • the method further comprises treating the subject with an anti-HER2 antibody, preferably rhuMAb 2C4.
  • the invention provides an article of manufacture comprising a container comprising an antibody which binds HER2 and instructions for administering the antibody to a patient suffering from a tumor.
  • the tumor has been determined to comprise HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 heterodimers.
  • the container comprises an antibody that blocks ligand activation of an ErbB heterodimer comprising HER2.
  • the container comprises monoclonal antibody 2C4, more preferably rhuMAb 2C4.
  • the invention provides a method of treating a patient comprising administering to the patient a therapeutically effective amount of an antibody which binds HER2.
  • the patient is suffering from a tumor which has been determined to comprise HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 heterodimers.
  • the antibody blocks the ligand activation of an ErbB heterodimer comprising HER2.
  • the antibody is monoclonal antibody 2C4, more preferably rhuMAb 2C4.
  • the invention provides a method of treating a patient comprising administering to the patient a therapeutically effective amount of an antibody which binds HER2.
  • the patient is suffering from a tumor which has been determined to have a phosphorylated ErbB receptor.
  • the phosphorylated ErbB receptor is HER2.
  • the antibody blocks the ligand activation of an ErbB heterodimer comprising HER2.
  • the antibody is monoclonal antibody 2C3, more preferably rhuMAb 2C4.
  • the present invention is based, in part, on the experimental finding that responsiveness to the anti-HER2 antibody rhuMAb 2C4 correlates with the presence of HER2/HER3 and/or HER2/HER1 and or HER2/HER4 heterodimers, and/or the phosphorylation of an ErbB receptor in tumor cells.
  • a tumor may be identified as responsive to treatment with an anti-HER2 antibody, particularly an anti-HER2 antibody that has one or more of the biological activities of the anti-HER2 antibody 2C4, based on the presence of HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 heterodimers, and/or the phosphorylation of an ErbB receptor.
  • HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 heterodimers, and/or ErbB receptor phosphorylation may be identified by any method known in the art. By identifying specific tumors and tumor types that are responsive to treatment with anti-HER2 antibodies, patients can be identified who will likely benefit the most from such treatment. In addition, patients that would likely not benefit from therapy with monoclonal antibody 2C4 can be identified.
  • ErbB receptor is a receptor protein tyrosine kinase which belongs to the ErbB receptor family and includes EGFR (ErbB1), ERRP, ErbB2, ErbB3 and ErbB4 receptors and other members of this family to be identified in the future.
  • the ErbB receptor will generally comprise an extracellular domain, which may bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the ErbB receptor may be a "native sequence" ErbB receptor or an "amino acid sequence variant" thereof.
  • the ErbB receptor is native sequence human ErbB receptor.
  • a "member of the ErbB receptor family" is EGFR (ErbB1), ErbB2, ErbB3, ErbB4 or any other ErbB receptor currently known or to be identified in the future.
  • the member is EGFR (ErbB1), ErbB2, ErbB3, or ErbB4.
  • ErbB1 "epidermal growth factor receptor", “EGFR” and “HER1” are used interchangeably herein and refer to EGFR as disclosed, for example, in Carpenter et al., Ann. Rev. Biochem., 56:881-914 (1987 ), including naturally occurring mutant forms thereof (e.g ., a deletion mutant EGFR as in Humphrey et al., PNAS (USA), 87:4207-4211 (1990 )).
  • erbB1 refers to the gene encoding the EGFR protein product.
  • Antibodies against HER1 are described, for example, in Murthy et al., Arch. Biochem. Biophys., 252:549-560 (1987 ) and in WO 95/25167 .
  • ERRP epidermal growth factor receptor
  • EGF epidermal growth factor receptor
  • ErbB2 and "HER2” are used interchangeably herein and refer to human HER2 protein described, for example, in Semba et al., PNAS (USA), 82:6497-6501 (1985 ) and Yamamoto et al., Nature, 319:230-234 (1986 ) (Genebank accession number X03363).
  • the term “erbB2” refers to the gene encoding human ErbB2 and "neu” refers to the gene encoding rat p 185neu.
  • Preferred ErbB2 is native sequence human ErbB2.
  • ErbB3 and "HER3” refer to the receptor polypeptide as disclosed, for example, in U.S. Patent Nos. 5,183,884 and 5,480,968 as well as Kraus et al., PNAS (USA), 86:9193-9197 (1989 ).
  • Antibodies against ErbB3 are known in the art and are described, for example, in U.S. Patent Nos. 5,183,884 , 5,480,968 and in WO 97/35885 .
  • ErbB4 and "HER4" herein refer to the receptor polypeptide as disclosed, for example, in EP Pat Appln No 599,274 ; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993 ); and Plowman et al., Nature, 366:473-475 (1993 ), including isoforms thereof, e.g ., as disclosed in WO 99/19488, published April 22, 1999 .
  • Antibodies against HER4 are described, for example, in WO 02/18444 .
  • Antibodies to ErbB receptors are available commercially from a number of sources, including, for example, Santa Cruz Biotechnology, Inc., California, USA.
  • ErbB ligand is meant a polypeptide which binds to and/or activates an ErbB receptor.
  • the ErbB ligand may be a native sequence human ErbB ligand such as epidermal growth factor (EGF) ( Savage et al., J. Biol.
  • TGF- ⁇ transforming growth factor alpha
  • amphiregulin also known as schwanoma or keratinocyte autocrine growth factor
  • Shoyab et al. Science, 243:1074-1076 (1989 ); Kimura et al., Nature, 348:257-260 (1990 ); and Cook et al., Mol. Cell. Biol., 11:2547-2557 (1991 )
  • betacellulin Shing et al., Science, 259:1604-1607 (1993 ); and Sasada et al., Biochem.
  • HB-EGF heparin-binding epidermal growth factor
  • epiregulin Toyoda et al., J. Biol. Chem., 270:7495-7500 (1995 ); and Komurasaki et al., Oncogene, 15:2841-2848 (1997 )
  • a heregulin see below
  • neuregulin-2 (NRG-2) ( Carraway et al., Nature, 387:512-516 (1997 )
  • neuregulin-3 (NRG-3) ( Zhang et al., Proc.
  • ErbB ligands which bind EGFR include EGF, TGF- ⁇ , amphiregulin, betacellulin, HB-EGF and epiregulin. ErbB ligands which bind ErbB3 include heregulins.
  • ErbB ligands capable of binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and heregulins.
  • the ErbB ligand may also be a synthetic ErbB ligand.
  • the synthetic ligand may be specific for a particular ErbB receptor, or may recognize particular ErbB receptor complexes.
  • An example of a synthetic ligand is the synthetic heregulin/egf chimera biregulin (see, for example, Jones et al., FEBS Letters, 447:227-231 (1999 ), which is incorporated by reference).
  • Heregulin when used herein refers to a polypeptide encoded by the heregulin gene product as disclosed in U.S. Patent No. 5,641,869 or Marchionni et al., Nature, 362:312-318 (1993 ).
  • Examples of heregulins include heregulin- ⁇ heregulin- ⁇ 1, heregulin- ⁇ 2 and heregulin- ⁇ 3 ( Holmes et al., Science, 256:1205-1210 (1992 ); and U.S. Patent No.
  • NDF neu differentiation factor
  • ARIA acetylcholine receptor-inducing activity
  • GGFs glial growth factors
  • SMDF sensory and motor neuron derived factor
  • the term includes biologically active fragments and/or amino acid sequence variants of a native sequence HRG polypeptide, such as an EGF-like domain fragment thereof ( e.g ., HRG ⁇ 1177-244).
  • ErbB hetero-oligomer herein is a noncovalently associated oligomer comprising at least two different ErbB receptors.
  • An "ErbB dimer” is a noncovalently associated oligomer that comprises two different ErbB receptors. Such complexes may form when a cell expressing two or more ErbB receptors is exposed to an ErbB ligand.
  • ErbB oligomers, such as ErbB dimers can be isolated by immunoprecipitation and analyzed by SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994 ), for example.
  • ErbB hetero-oligomers examples include EGFR-ErbB2 (also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes.
  • the ErbB hetero-oligomer may comprise two or more ErbB2 receptors combined with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR (ErbB1).
  • Other proteins, such as a cytokine receptor subunit e.g ., gp 130
  • a cytokine receptor subunit e.g ., gp 130
  • ligand activation of an ErbB receptor is meant signal transduction (e.g ., that caused by an intracellular kinase domain of an ErbB receptor phosphorylating tyrosine residues in the ErbB receptor or a substrate polypeptide) mediated by ErbB ligand binding to a ErbB hetero-oligomer comprising the ErbB receptor of interest.
  • this will involve binding of an ErbB ligand to an ErbB hetero-oligomer which activates a kinase domain of one or more of the ErbB receptors in the hetero-oligomer and thereby results in phosphorylation of tyrosine residues in one or more of the ErbB receptors and/or phosphorylation of tyrosine residues in additional substrate polypeptides(s).
  • ErbB receptor activation can be quantified using various tyrosine phosphorylation assays.
  • a “native sequence” polypeptide is one which has the same amino acid sequence as a polypeptide (e.g ., ErbB receptor or ErbB ligand) derived from nature.
  • a polypeptide e.g ., ErbB receptor or ErbB ligand
  • Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • a native sequence polypeptide can have the amino acid sequence of naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species.
  • amino acid sequence variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% homology with at least one receptor binding domain of a native ErbB ligand or with at least one ligand binding domain of a native ErbB receptor, and preferably, they will be at least about 80%, more preferably, at least about 90% homologous with such receptor or ligand binding domains. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
  • “Homology” is defined as the percentage of residues in the amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. One such computer program is "Align 2,” authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, DC 20559, on December 10, 1991.
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g ., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i . e ., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975 ), or may be made by recombinant DNA methods (see, e.g ., U.S. Patent No. 4,816,567 ).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., J. Mol. Biol., 222:581-597 (1991 ), for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity ( U.S. Patent No. 4,816,567 ; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984 )).
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g ., Old World Monkey, Ape etc) and human constant region sequences.
  • Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).
  • an “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.
  • the constant domains may be native sequence constant domains (e.g ., human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody.
  • Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g ., B cell receptor; BCR), etc.
  • intact antibodies can be assigned to different "classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g ., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy-chain constant domains that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g ., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs Fc receptors
  • FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991 ).
  • ADCC activity of a molecule of interest may be assessed in vitro, such as that described in US Patent No. 5,500,362 or 5,821,337.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g ., in a animal model such as that disclosed in Clynes et al., PNAS (USA), 95:652-656 (1998 ).
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc ⁇ RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells and neutrophils
  • the effector cells may be isolated from a native source thereof, e.g ., from blood or PBMCs as described herein.
  • Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor") and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR FcR
  • FcRn neonatal receptor
  • “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g ., an antibody) complexed with a cognate antigen.
  • a CDC assay e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996 ), may be performed.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region” or "CDR" (e.g ., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • CDR complementarity determining region
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda (X), based on the amino acid sequences of their constant domains.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • scFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994 ).
  • Anti-ErbB2 antibody scFv fragments are described in WO 93/16185 ; U.S. Patent No. 5,571,894 ; and U.S. Patent No. 5,587,458 .
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH - VL).
  • VH variable heavy domain
  • VL variable light domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097 ; WO 93/11161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993 ).
  • Humanized forms of non-human (e.g ., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as described in Table 3 of U.S. Patent No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 ( WO 93/21319 ) and humanized 2C4 antibodies as described herein below.
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • an antibody "which binds" an antigen of interest e.g ., ErbB2 antigen
  • an antigen of interest e.g ., ErbB2 antigen
  • an antigen of interest e.g ., ErbB2 antigen
  • the extent of binding of the antibody to these non-ErbB2 proteins will be less than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).
  • FACS fluorescence activated cell sorting
  • RIA radioimmunoprecipitation
  • the anti-ErbB2 antibody will not significantly cross-react with the rat neu protein, e.g ., as described in Schecter et al., Nature, 312:513 (1984 ) and Drebin et al., Nature, 312:545-548 (1984 ).
  • an antibody which "blocks" ligand activation of an ErbB receptor is one which reduces or prevents such activation as hereinabove defined, wherein the antibody is able to block ligand activation of the ErbB receptor substantially more effectively than monoclonal antibody 4D5, e.g ., about as effectively as monoclonal antibodies 7F3 or 2C4 or Fab fragments thereof and preferably about as effectively as monoclonal antibody 2C4 or a Fab fragment thereof.
  • the antibody that blocks ligand activation of an ErbB receptor may be one which is about 50-100% more effective than 4D5 at blocking formation of an ErbB hetero-oligomer.
  • Blocking of ligand activation of an ErbB receptor can occur by any means, e.g ., by interfering with: ligand binding to an ErbB receptor, ErbB complex formation, tyrosine kinase activity of an ErbB receptor in an ErbB complex and/or phosphorylation of tyrosine kinase residue(s) in or by an ErbB receptor.
  • Examples of antibodies which block ligand activation of an ErbB receptor include monoclonal antibodies 2C4 and 7F3 (which block HRG activation ofErbB2/ErbB3 and ErbB2/ErbB4 hetero-oligomers; and EGF, TGF- ⁇ , amphiregulin, HB-EGF and/or epiregulin activation of an EGFR/ErbB2 hetero-oligomer); and L26, L96 and L288 antibodies ( Klapper et al., Oncogene, 14:2099-2109 (1997 )), which block EGF and NDF binding to T47D cells which express EGFR, ErbB2, ErbB3 and ErbB4.
  • An antibody having a "biological characteristic" of a designated antibody such as the monoclonal antibody designated 2C4, is one which possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies that bind to the same antigen (e.g ., ErbB2).
  • an antibody with a biological characteristic of 2C4 may block HRG activation of an ErbB hetero-oligomer comprising ErbB2 and ErbB3, ErbB1 or ErbB4; block EGF, TGF- ⁇ , HB-EGF, epiregulin and/or amphiregulin activation of an ErbB receptor comprising EGFR and ErbB2; block EGF, TGF- ⁇ and/or HRG mediated activation of MAPK; and/or bind the same epitope in the extracellular domain of ErbB2 as that bound by 2C4 ( e.g ., which blocks binding of monoclonal antibody 2C4 to ErbB2).
  • the expression “monoclonal antibody 2C4" refers to an antibody that has antigen binding residues of, or derived from, the murine 2C4 antibody of the Examples below.
  • the monoclonal antibody 2C4 may be murine monoclonal antibody 2C4 or a variant thereof, such as humanized antibody 2C4, possessing antigen binding amino acid residues of murine monoclonal antibody 2C4.
  • humanized 2C4 antibodies are provided herein and in WO 01/00245 , which is incorporated herein by reference in its entirety.
  • the expression “rhuMAb 2C4" when used herein refers to an antibody comprising the variable light (VL) and variable heavy (VH) sequences of SEQ ID Nos. 3 and 4, respectively, fused to human light and heavy IgG1 (non-A allotype) constant region sequences optionally expressed by a Chinese Hamster Ovary (CHO) cell.
  • the term “monoclonal antibody 4D5" refers to an antibody that has antigen binding residues of, or derived from, the murine 4D5 antibody (ATCC CRL 10463).
  • the monoclonal antibody 4D5 may be murine monoclonal antibody 4D5 or a variant thereof, such as a humanized 4D5, possessing antigen binding residues of murine monoclonal antibody 4D5.
  • Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (HERCEPTIN®) as in US Patent No. 5,821,337 , with huMAb4D5-8 (HERCEPTIN®) being a preferred humanized 4D5 antibody.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially an ErbB expressing cancer cell either in vitro or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of ErbB expressing cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • growth inhibitory antibodies are those which bind to ErbB2 and inhibit the growth of cancer cells overexpressing ErbB2.
  • Preferred growth inhibitory anti-ErbB2 antibodies inhibit growth of SK-BR-3 breast tumor cells in cell culture by greater than 20%, and preferably greater than 50% ( e.g ., from about 50% to about 100%) at an antibody concentration of about 0.5 to 30 ⁇ g/ml, where the growth inhibition is determined six days after exposure of the SK-BR-3 cells to the antibody (see U.S. Patent No. 5,677,171 issued October 14, 1997 ).
  • the SK-BR-3 cell growth inhibition assay is described in more detail in that patent and herein below.
  • the preferred growth inhibitory antibody is monoclonal antibody 4D5, e.g ., humanized 4D5.
  • the cell is generally one which expresses the ErbB2 receptor, especially where the cell overexpresses the ErbB2 receptor.
  • the cell is a cancer cell, e.g ., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell.
  • the cell may be a SK-BR-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell.
  • Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • the assay for cell death may be performed using heat inactivated serum (i.e ., in the absence of complement) and in the absence of immune effector cells.
  • PI propidium iodide
  • trypan blue see Moore et al., Cytotechnology, 17:1-11 (1995 )
  • 7AAD can be assessed relative to untreated cells.
  • Preferred cell death-inducing antibodies are those which induce PI uptake in the PI uptake assay in BT474 cells (see below).
  • an antibody which "induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
  • the cell is usually one which overexpresses the ErbB2 receptor.
  • the cell is a tumor cell, e.g ., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell.
  • the cell maybe a SK-BR-3, BT474, Calu 3 cell, MDA-MB-453, MDA-MB-361 or SKOV3 cell.
  • Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.
  • PS phosphatidyl serine
  • the antibody which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay using BT474 cells (see below).
  • the pro-apoptotic antibody will be one which further blocks ErbB ligand activation of an ErbB receptor (e.g ., 7F3 antibody); i.e., the antibody shares a biological characteristic with monoclonal antibody 2C4. In other situations, the antibody is one which does not significantly block ErbB ligand activation of an ErbB receptor ( e.g ., 7C2).
  • the antibody may be one like 7C2 which, while inducing apoptosis, does not induce a large reduction in the percent of cells in S phase ( e.g ., one which only induces about 0-10% reduction in the percent of these cells relative to control).
  • the "epitope 2C4" is the region in the extracellular domain of ErbB2 to which the antibody 2C4 binds.
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988 ), can be performed.
  • epitope mapping can be performed to assess whether the antibody binds to the 2C4 epitope of ErbB2 ( e.g ., any one or more residues in the region from about residue 22 to about residue 584 of ErbB2, inclusive; see Figs. 1A-B ).
  • the "epitope 4D5" is the region in the extracellular domain of ErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds. This epitope is close to the transmembrane domain of ErbB2.
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988 ), can be performed.
  • epitope mapping can be performed to assess whether the antibody binds to the 4D5 epitope of ErbB2 (e.g ., any one or more residues in the region from about residue 529 to about residue 625, inclusive; see Figs. 1A-B ).
  • the “epitope 3H4" is the region in the extracellular domain of ErbB2 to which the antibody 3H4 binds. This epitope includes residues from about 541 to about 599, inclusive, in the amino acid sequence of ErbB2 extracellular domain; see Figs. 1A-B .
  • the “epitope 7C2/7F3” is the region at the N terminus of the extracellular domain of ErbB2 to which the 7C2 and/or 7F3 antibodies (each deposited with the ATCC, see below) bind.
  • a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988 ), can be performed.
  • epitope mapping can be performed to establish whether the antibody binds to the 7C2/7F3 epitope on ErbB2 (e.g ., any one or more of residues in the region from about residue 22 to about residue 53 of ErbB2; see Figs. 1A-B ).
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
  • a tumor which is "responsive to treatment” shows statistically significant improvement in response to anti-ErbB antibody treatment when compared to no treatment or treatment with placebo in a recognized animal model or a human clinical trial, or which responds to initial treatment with anti-ErbB antibodies but grows as treatment is continued.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized ( i . e ., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • a “disorder” is any condition that would benefit from treatment of the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • disorders to be treated herein include benign and malignant tumors; leukemias and lymphoid malignancies, in particular breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.
  • a preferred disorder to be treated in accordance with the present invention is malignant tumors, leukemias and lymphoid malignancies, in particular breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer; neuronal, glial, astrocytal, hypo
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit ( i.e ., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit ( i.e ., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g ., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer ("NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • NSCLC non-small cell lung cancer
  • adenocarcinoma of the lung and squamous carcinoma of the lung cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including
  • An "ErbB-expressing cancer” is one comprising cells which have ErbB protein present at their cell surface.
  • An "ErbB2-expressing cancer” is one which produces sufficient levels of ErbB2 at the surface of cells thereof, such that an anti-ErbB2 antibody can bind thereto and have a therapeutic effect with respect to the cancer.
  • a cancer "characterized by excessive activation" of an ErbB receptor is one in which the extent of ErbB receptor activation in cancer cells significantly exceeds the level of activation of that receptor in non-cancerous cells of the same tissue type. Such excessive activation may result from overexpression of the ErbB receptor and/or greater than normal levels of an ErbB ligand available for activating the ErbB receptor in the cancer cells. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of an ErbB receptor is occurring which results in such excessive activation of the ErbB receptor.
  • the cancer may be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB ligand is occurring in the cancer which attributes to excessive activation of the receptor.
  • a diagnostic or prognostic assay to determine whether amplification and/or overexpression an ErbB ligand is occurring in the cancer which attributes to excessive activation of the receptor.
  • excessive activation of the receptor may result from an autocrine stimulatory pathway.
  • the cancer may express or overexpress EGFR and also express or overexpress an EGFR ligand (e.g ., EGF, TGF- ⁇ , or HB-EGF).
  • the cancer may express or overexpress ErbB2 and also express or overexpress a heregulin (e.g. ⁇ -HRG).
  • a cancer which "overexpresses" an ErbB receptor is one which has significantly higher levels of an ErbB receptor, such as ErbB2, at the cell surface thereof, compared to a noncancerous cell of the same tissue type.
  • Such overexpression may be caused by gene amplification or by increased transcription or translation.
  • ErbB receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the ErbB protein present on the surface of a cell ( e.g ., via an immunohistochemistry assay; IHC).
  • FISH fluorescent in situ hybridization
  • PCR polymerase chain reaction
  • Overexpression of the ErbB ligand may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g ., in a tumor biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR or in vivo assays described above.
  • a detectable label e.g ., a radioactive isotope
  • 0 0-10,000 copies/cell
  • 1+ at least about 200,000 copies/cell
  • 2+ at least about 500,000 copies/cell
  • 3+ at least about 2,000,000 copies/cell.
  • Overexpression of HER2 at the 3+ level which leads to ligand-independent activation of the tyrosine kinase ( Hudziak et al., Proc. Natl. Acad. Sci.
  • a cancer which is "not characterized by overexpression of the ErbB2 receptor" is one which, in a diagnostic assay, does not express higher than normal levels of ErbB2 receptor compared to a noncancerous cell of the same tissue type.
  • a “hormone independent” cancer is one in which proliferation thereof is not dependent on the presence of a hormone which binds to a receptor expressed by cells in the cancer. Such cancers do not undergo clinical regression upon administration of pharmacological or surgical strategies that reduce the hormone concentration in or near the tumor.
  • hormone independent cancers include androgen independent prostate cancer, estrogen independent breast cancer, endometrial cancer and ovarian cancer. Such cancers may begin as hormone dependent tumors and progress from a hormone-sensitive stage to a hormone-refractory tumor following anti-hormonal therapy.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofos
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • antiestrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • EGFR-targeted drug refers to a therapeutic agent that binds to EGFR and, optionally, inhibits EGFR activation.
  • agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No.
  • the anti-EGFR antibody may be conjugated with a cyotoxic agent, thus generating an immunoconjugate (see, e.g ., EP 659,439A2 , Merck Patent GmbH).
  • a cyotoxic agent see, e.g ., EP 659,439A2 , Merck Patent GmbH.
  • small molecules that bind to EGFR include ZD1839 or Gefitinib (IRESSATM; Astra Zeneca), CP-358774 (TARCEVATM; Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).
  • a "tyrosine kinase inhibitor” is a molecule which inhibits to some extent tyrosine kinase activity of a tyrosine kinase such as an ErbB receptor.
  • inhibitors include the EGFR-targeted drugs noted in the preceding paragraph as well as quinazolines such as PD 153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines, curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide), tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g ., those that bind to ErbB-encoding nucleic acid); quinoxalines ( U.S.
  • quinazolines such as PD 153035,4-(
  • Patent No. 5,804,396 tryphostins ( U.S. Patent No. 5,804,396 ); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as described in any of the following patent publications: U.S.
  • Patent No. 5,804,396 WO 99/09016 (American Cyanimid); WO 98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca).
  • an "anti-angiogenic agent” refers to a compound which blocks, or interferes with to some degree, the development of blood vessels.
  • the anti-angiogenic factor may, for instance, be a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.
  • the preferred anti-angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF).
  • VEGF Vascular Endothelial Growth Factor
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF- ⁇ ; platelet-growth factor;
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g ., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382 , 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985 ).
  • the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
  • a drug such as the anti-ErbB2 antibodies disclosed herein and, optionally, a chemotherapeutic agent
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • a “cardioprotectant” is a compound or composition which prevents or reduces myocardial dysfunction (i . e ., cardiomyopathy and/or congestive heart failure) associated with administration of a drug, such as an anthracycline antibiotic and/or an anti-ErbB2 antibody, to a patient.
  • the cardioprotectant may, for example, block or reduce a free-radical-mediated cardiotoxic effect and/or prevent or reduce oxidative-stress injury.
  • cardioprotectants encompassed by the present definition include the iron-chelating agent dexrazoxane (ICRF-187) ( Seifert et al., The Annals of Pharmacotherapy, 28:1063-1072 (1994 )); a lipid-lowering agent and/or anti-oxidant such as probucol ( Singal et al., J. Mol.
  • amifostine (aminothiol 2-[(3-aminopropyl)amino]ethanethiol-dihydrogen phosphate ester, also called WR-2721, and the dephosphorylated cellular uptake form thereof called WR-1065) and S-3-(3-methylaminopropylamino)propylphosphorothioic acid (WR-151327), see Green et al., Cancer Research, 54:738-741 (1994 ); digoxin ( Bristow, M.R. In: Bristow MR, ed. Drug-Induced Heart Disease.
  • beta-blockers such as metoprolol ( Hjalmarson et al., Drugs 47:Suppl 4:31-9 (1994 ); and Shaddy et al., Am.
  • vitamin E ascorbic acid (vitamin C); free radical scavengers such as oleanolic acid, ursolic acid and N-acetylcysteine (NAC); spin trapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN); ( Paracchini et al., Anticancer Res., 13:1607-1612 (1993 )); selenoorganic compounds such as P251 (Elbesen); and the like.
  • vitamin C ascorbic acid
  • free radical scavengers such as oleanolic acid, ursolic acid and N-acetylcysteine (NAC)
  • spin trapping compounds such as alpha-phenyl-tert-butyl nitrone (PBN); ( Paracchini et al., Anticancer Res., 13:1607-1612 (1993 )); selenoorganic compounds such as P251 (Elbesen); and the like.
  • an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • Tumors can be characterized as responsive to therapy with 2C4, or functionally equivalent antibodies, that is, antibodies having one or more of the biological characteristics of antibody 2C4, e.g ., based on the presence of EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers on the cell surface, as a measure of HER2 activation.
  • Tumor samples may be assayed for the presence of heterodimers by any method known in the art.
  • the presence of heterodimers is determined by one or more of the methods described below.
  • HER2 activation is the result of receptor heterodimerization and phosphorylation
  • a particularly important method for identifying tumors responsive to therapy with 2C4, or functionally equivalent antibodies is the detection of phosphorylation of ErbB receptor, such as phosphorylation of ErbB2 (HER2) receptor, as described below.
  • Sources of tumor cells that may be assayed include, but are not limited to, tumor biopsies, circulating tumor cells, circulating plasma proteins, ascitic fluid, xenotransplanted tumors and other tumor models, and primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, as well as preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples.
  • the screening of panels of various tumor cell types for EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers, and/or phosphorylation of ErbB receptor is contemplated by the present invention.
  • Tumor cells of the same type as tumor cells that test positive for heterodimers, and/or phosphorylation of ErbB receptor, such as ErbB2 (HER2) receptor, may be subjected to therapy with 2C4.
  • ErbB2 ErbB2
  • the tumor models described below are provided as examples and should not be construed as limiting the invention.
  • tumor cells that originate with a patient currently suffering from a tumor are assayed for responsiveness to therapy with 2C4. If the cells are determined to be responsive, based on the presence of HER2/HER3 and/or HER2/HER1 heterodimers or by demonstrating the phosphorylation of ErbB receptor, the patient may subsequently be treated with an antibody with one or more of the biological characteristics of 2C4. Preferably, the patient is treated with rhuMAb 2C4.
  • tumor cells from particular type of tumor or cells that are believed to have the characteristics of a particular type of tumor are assayed for the presence of EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers, or for the phosphorylation of ErbB receptor, preferable ErbB2 (HER2) receptor. If EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers and/or phosphorylation of ErbB receptor is detected, that type of tumor is considered to be a good candidate for treatment with an anti-ErbB2 antibody with one or more of the biological characteristics of 2C4. Patients suffering from that type of tumor may then be treated with such an antibody.
  • tumor cells such as tumor cells grown in culture and tumor cell lines
  • Such methods are well known to one of skill in the art.
  • the cells are assayed to identify the presence of EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers, or for the phosphorylation of ErbB receptor, such as the phosphorylation of ErbB2 (HER2) receptor.
  • HER2 phosphorylation of ErbB2
  • tumor cells are implanted subcutaneously into a mouse, preferably an athymic nude mouse.
  • tumor cells are implanted into a physiologically relevant location to create an appropriate in situ tumor model.
  • cells from a breast cancer cell line may be implanted at various concentrations into the mammary fat pad of athymic nude mice to more accurately model the biology of breast cancer.
  • Tumor cells may be assayed for the presence of EGFR-ErbB2 or ErbB2-ErbB3 heterodimers, or for the phosphorylation of ErbB receptor either before or after implantation.
  • tumor cells are assayed after the implanted cells have developed into a tumor of a predetermined size.
  • the mice may also be subjected to therapy with 2C4 or a functionally equivalent antibody, with untreated mice serving as a control.
  • Tumor types include, but are not limited to, bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, melanoma, ovary, pancreas, prostate, sarcoma, stomach, testicle, and uterus.
  • tumor cells or cell lines that overexpress ErbB2 are used for implantation, while in another embodiment, tumor cells or cell lines that express normal or below normal amounts of ErbB2 are used for implantation.
  • tumor cells or cell lines non-responsive to HERCEPTIN® are used for implantation.
  • approximately 20 million MDA-175 breast tumor cells are implanted into the mouse gonadal fat pad.
  • Expression of HER2/HER1 and/or HER2/HER3 dimers on the surface of xenografted cells is determined, such as by one of the methods described below.
  • Mice thus implanted may also be subjected to treatment with 0, 3 mg/kg, 10 mg/kg, 30 mg/kg, or 100 mg/kg 2C4.
  • Other dosage regimens would be within the determination of one of ordinary skill in the art.
  • the present invention is suitable for the classification of any HER2 expressing tumor, solid tumors, like breast cancer, ovarian cancer, lung cancer, prostate cancer and colorectal cancer, are particularly suitable for analysis and treatment following the present invention.
  • Mammalian tumor specimens preferably human tumor specimens
  • the tumor specimens may be obtained by any method known in the art.
  • the tumor specimens are surgically resected, such as in a biopsy or in the process of surgery to remove the tumor from the mammal.
  • the tumor specimen is obtained by purifying circulating tumor cells from the mammals blood.
  • solid human tumor slices of approximately 5 x 5 x 0.5 to 1 mm in diameter are implanted into the flanks of athymic nude mice, generally four fragments per mouse. When the implanted tumors reach a median diameter of about 10-15 mm, they may be serially passaged, generally using smaller tumor fragments.
  • Methods of generating and studying human tumor xenografts are described in the following references, herein incorporated in their entirety: Fiebig et al., "Human Tumor Xenografts: Predictivity, Characterization and Discovery of New Anticancer Agents," in Contributions to Oncology: Relevance of Tumor Models for Anticancer Drug Development, Fiebig & Burger, eds.
  • xenografts are considered highly predictive of tumor behavior within the donor patient, as the xenograft grows as a solid tumor, differentiates, and develops a stroma, vasculature, and a central necrosis. In most cases, xenografts retain most of the molecular, histological, and pathophysiological characteristics of the fresh patient-derived tumor.
  • Tumor cells from mice containing first or serially passaged tumors may be analyzed for the presence of EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers, or for the phosphorylation of ErbB receptor. Mice may also be subjected to therapy with 2C4 or a functionally equivalent antibody.
  • a newly created or established panel of human tumor xenografts is screened for the presence of EGFR-ErbB2 or ErbB2-ErbB3 heterodimers, or for phosphorylation of ErbB receptor.
  • Fiebig & Burger, supra describe a panel of over 300 human tumor xenografts established from a variety of common cancer types, such as bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, melanoma, ovary, pancreas, prostate, sarcoma, stomach, testicle, and uterus.
  • the entire panel is screened for heterodimers, or for phosphorylation of ErbB receptor, such as ErbB2 (HER2) receptor.
  • Subsets of this panel may also be screened for heterodimers, or for phosphorylation of ErbB receptor, wherein subsets are based on, for example, tissue type, over-, under-, or normal expression of ErbB2, or failure to respond to HERCEPTIN®.
  • tumors may be categorized as candidates for therapy with 2C4 based on the presence of heterodimers, or by demonstrating the phosphorylation of ErbB receptor, such as ErbB2 (HER2) receptor.
  • patients possessing tumors thus categorized may be more rapidly deemed eligible for therapy with 2C4 or a functionally equivalent antibody.
  • Tumor specimens may be assayed for the presence of EGFR-ErbB2 or ErbB2-ErbB3 heterodimers, or for the phosphorylation of ErbB receptor either before or after implantation.
  • approximately one gram of tumor from a first and/or serially passaged xenograft is further characterized molecularly for heterodimers or snap frozen in liquid nitrogen and stored at -80°C for later characterization.
  • Xenograft tumors may be further analyzed by a double layer soft-agarassay, also called a clonogenic assay, as described, for example, in Fiebig & Burger, supra.
  • Solid human tumor xenografts are mechanically and proteolytically disaggregated into a single-cell suspension, which is plated into multiwell plates layered with soft agar as described. Tumor cell growth in vitro leads to the formation of colonies, which may be further analyzed for molecular characteristics, such as heterodimers, or for phosphorylation of ErbB receptor, or for other histochemical or morphological characteristics.
  • Any method known in the art may be used to detect EGFR-ErbB2 or ErbB2-ErbB3 heterodimers in tumors. Several preferred methods are described below. These methods detect noncovalent protein-protein interactions or otherwise indicate proximity between proteins of interest. The methods described below are provided as examples and should not be construed as limiting the invention.
  • Immunoaffinity-based methods may be used to detect EGFR-ErbB2 or ErbB2-ErbB3 heterodimers.
  • anti-ErbB2 antibodies are used to immunoprecipitate complexes comprising ErbB2 from tumor cells, and the resulting immunoprecipitant is then probed for the presence of EGFR or ErbB3 by immunoblotting.
  • EGFR or ErbB3 antibodies may be used for the immunoprecipitation step and the immunoprecipitant then probed with ErbB2 antibodies.
  • ErbB ligands specific to HER1, HER3, HER2/HER1 complexes or HER2/HER3 complexes may be used to precipitate complexes, which are then probed for the presence of HER2.
  • ligands may be conjugated to avidin and complexes purified on a biotin column.
  • antibodies to ErbB2 are immobilized on a solid support, contacted with tumor cells or tumor cell lysate, washed, and then exposed to antibody against EGFR or ErbB3. Binding of the latter antibody, which may be detected directly or by a secondary antibody conjugated to a detectable label, indicates the presence of heterodimers.
  • EGFR or ErbB3 antibody is immobilized, and ErbB2 antibody is used for the detection step.
  • ErbB receptor ligands may be used in place of, or in combination with anti-ErbB receptor antibodies.
  • Immunoprecipitation with EGFR, ErbB2, or ErbB3 antibody may be followed by a functional assay for heterodimers, as an alternative or supplement to immunoblotting.
  • immunoprecipitation with ErbB3 antibody is followed by an assay for receptor tyrosine kinase activity in the immunoprecipitant. Because ErbB3 does not have intrinsic tyrosine kinase activity, the presence of tyrosine kinase activity in the immunoprecipitant indicates that ErbB3 is most likely associated with ErbB2.
  • immunoprecipitation with ErbB2 antibody is followed by an assay for EGFR receptor tyrosine kinase activity.
  • the immunoprecipitant is contacted with radioactive ATP and a peptide substrate that mimics the in vivo site of transphosphorylation of ErbB2 by EGFR. Phosphorylation of the peptide indicates co-immunoprecipitation and thus heterodimerization of EGFR with ErbB2.
  • Receptor tyrosine kinase activity assays are well known in the art and include assays that detect phosphorylation of target substrates, for example, by phosphotyrosine antibody, and activation of cognate signal transduction pathways, such as the MAPK pathway.
  • Chemical or UV cross-linking may also be used to covalently join heterodimers on the surface of living cells. Hunter et al., Biochem. J., 320:847-53 .
  • chemical cross-linkers include dithiobis(succinimidyl) propionate (DSP) and 3,3'dithiobis(sulphosuccinimidyl) propionate (DTSSP).
  • DSP dithiobis(succinimidyl) propionate
  • DTSSP 3,3'dithiobis(sulphosuccinimidyl) propionate
  • cell extracts from chemically cross-linked tumor cells are analyzed by SDS-PAGE and immunoblotted with antibodies to EGFR and/or ErbB3.
  • a supershifted band of the appropriate molecular weight most likely represents EGFR-ErbB2 or ErbB2-ErbB3 heterodimers, as ErbB2 is the preferred heterodimerization partner for EGFR and ErbB3. This result may be confirmed by subsequent immunoblotting with ErbB2 antibodies.
  • Fluorescence resonance energy transfer may also be used to detect EGFR-ErbB2 or ErbB2-ErbB3 heterodimers.
  • FRET detects protein conformational changes and protein-protein interactions in vivo and in vitro based on the transfer of energy from a donor fluorophore to an acceptor fluorophore.
  • two proteins or two sites on a single protein are labeled with different fluorescent probes. One of the probes, the donor probe, is excited to a higher energy state by incident light of a specified wavelength.
  • the donor probe then transmits its energy to the second probe, the acceptor probe, resulting in a reduction in the donor's fluorescence intensity and an increase in the acceptor's fluorescence emission.
  • the donor's intensity in a sample labeled with donor and acceptor probes is compared with its intensity in a sample labeled with donor probe only.
  • acceptor intensity is compared in donor/acceptor and acceptor only samples.
  • Suitable probes are known in the art and include, for example, membrane permeant dyes, such as fluorescein and rhodamine, organic dyes, such as the cyanine dyes, and lanthanide atoms.
  • membrane permeant dyes such as fluorescein and rhodamine
  • organic dyes such as the cyanine dyes
  • lanthanide atoms such as the cyanine dyes
  • FRET-based techniques suitable for detecting and measuring protein-protein interactions in individual cells are also known in the art.
  • donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM) may be used to detect the dimerization of cell surface receptors.
  • pbFRET donor photobleaching fluorescence resonance energy transfer
  • FLIM fluorescence lifetime imaging microscopy
  • pbFRET is used on cells either "in suspension” or "in situ” to detect and measure the formation of EGFR-ErbB2 or ErbB2-ErbB3 heterodimers, as described in Nagy et al., Cytometry, 32:120-131 (1998 ).
  • FCET flow cytometric Foerster-type FRET technique
  • FRET is preferably used in conjunction with standard immunohistochemical labeling techniques. Kenworthy, Methods, 24:289-96 (2001 ).
  • antibodies conjugated to suitable fluorescent dyes can be used as probes for labeling two different proteins. If the proteins are within proximity of one another, the fluorescent dyes act as donors and acceptors for FRET.
  • Energy transfer is detected by standard means. Energy transfer may be detected by flow cytometric means or by digital microscopy systems, such as confocal microscopy or wide-field fluorescence microscopy coupled to a charge-coupled device (CCD) camera.
  • CCD charge-coupled device
  • ErbB2 antibodies and either EGFR or ErbB3 antibodies are directly labeled with two different fluorophores, for example as described in Nagy et al, supra.
  • Tumor cells or tumor cell lysates are contacted with the differentially labeled antibodies, which act as donors and acceptors for FRET in the presence of EGFR-ErbB2 or ErbB2-ErbB3 heterodimers.
  • unlabeled antibodies against ErbB2 and either EGFR or ErbB3 are used along with differentially labeled secondary antibodies that serve as donors and acceptors. See, for example, Brockhoff et al., supra. Energy transfer is detected and the presence of heterodimers is determined if the labels are found to be in close proximity.
  • ErbB receptor ligands that are specific for HER2 and either HER1 or HER3 are fluorescently labeled and used for FRET studies.
  • the presence of heterodimers on the surface of tumor cells is demonstrated by co-localization of ErbB2 with either EGFR or ErbB3 using standard direct or indirect immunofluorescence techniques and confocal laser scanning microscopy.
  • laser scanning imaging LSI is used to detect antibody binding and co-localization of ErbB2 with either EGFR or ErbB3 in a high-throughput format, such as a microwell plate, as described in Zuck et al, Proc. Natl. Acad. Sci. USA, 96:11122-27 (1999 ).
  • the presence of EGFR-ErbB2 and/or ErbB2-ErbB3 heterodimers is determined by identifying enzymatic activity that is dependent upon the proximity of the heterodimer components.
  • a ErbB2 antibody is conjugated with one enzyme and an EGFR or ErbB3 antibody is conjugated with a second enzyme.
  • a first substrate for the first enzyme is added and the reaction produces a second substrate for the second enzyme. This leads to a reaction with another molecule to produce a detectable compound, such as a dye.
  • the presence of another chemical breaks down the second substrate, so that reaction with the second enzyme is prevented unless the first and second enzymes, and thus the two antibodies, are in close proximity.
  • tumor cells or cell lysates are contacted with an ErbB2 antibody that is conjugated with glucose oxidase and an ErbB3 or ErbB1 antibody that is conjugated with horse radish peroxidase.
  • Glucose is added to the reaction, along with a dye precursor, such as DAB, and catalase. The presence of heterodimers is determined by the development of color upon staining for DAB.
  • Heterodimers may be detected using methods based on the eTagTM assay system (Aclara Bio Sciences, Mountain View, CA), as described, for example, WO 83502 and in U.S. Patent Application 2001/0049105, published December 6, 2001 , both of which are expressly incorporated by reference in their entirety.
  • An eTagTM, or "electrophoretic tag” comprises a detectable reporter moiety, such as a fluorescent group. It may also comprise a "mobility modifier,” which consists essentially of a moiety having a unique electrophoretic mobility. These moieties allow for separation and detection of the eTagTM from a complex mixture under defined electrophoretic conditions, such as capillary electrophoresis (CE).
  • CE capillary electrophoresis
  • the portion of the eTagTM containing the reporter moiety and, optionally, the mobility modifier is linked to a first target binding moiety by a cleavable linking group to produce a first binding compound.
  • the first target binding moiety specifically recognizes a particular first target, such as a nucleic acid or protein.
  • the first target binding moiety is not limited in any way, and may be for example, a polynucleotide or a polypeptide.
  • the first target binding moiety is an antibody or antibody fragment.
  • the first target binding moiety may be an ErbB receptor ligand or binding-competent fragment thereof.
  • the linking group preferably comprises a cleavable moiety, such as an enzyme substrate, or any chemical bond that may be cleaved under defined conditions.
  • a cleavable moiety such as an enzyme substrate, or any chemical bond that may be cleaved under defined conditions.
  • a second binding compound comprises the cleaving agent and a second target binding moiety that specifically recognizes a second target.
  • the second target binding moiety is also not limited in any way and may be, for example, an antibody or antibody fragment or an ErbB receptor ligand or binding competent ligand fragment.
  • the cleaving agent is such that it will only cleave the linking group in the first binding compound if the first binding compound and the second binding compound are in close proximity.
  • a first binding compound comprises an eTagTM in which an antibody to ErbB2 serves as the first target binding moiety.
  • a second binding compound comprises an antibody to EGFR or ErbB3 joined to a cleaving agent capable of cleaving the linking group of the eTagTM.
  • the cleaving agent must be activated in order to be able to cleave the linking group.
  • Tumor cells or tumor cell lysates are contacted with the eTagTM, which binds to ErbB2, and with the modified EGFR or ErbB3 antibody, which binds to EGFR or ErbB3 on the cell surface. Unbound binding compound is preferable removed, and the cleaving agent is activated, if necessary.
  • the cleaving agent will cleave the linking group and release the eTagTM due to the proximity of the cleaving agent to the linking group. Free eTagTM may then be detected by any method known in the art, such as capillary electrophoresis.
  • the cleaving agent is an activatable chemical species that acts on the linking group.
  • the cleaving agent may be activated by exposing the sample to light.
  • the eTagTM is constructed using an antibody to EGFR or ErbB3 as the first target binding moiety, and the second binding compound is constructed from an antibody to ErbB2.
  • the presence of the phosphorylation of ErbB receptor may be used to demonstrate HER2 activation.
  • phosphorylation of ErbB receptor is assessed by immunoprecipitation of one or more ErbB receptors, such as ErbB2 (HER2) receptor, and Western blot analysis. For example, positivity is determined by the presence of a phospho-HER2 band on the gel, using an anti-phosphotyrosine antibody to detect phosphorylated tyrosine residue(s) in the immunoprecipitated ErbB receptor(s).
  • Anti-phosphotyrosine antibodies are commercially available from PanVera (Madison, WI), a subsidiary of Invitrogen, Chemicon International Inc. (Temecula, CA), or Upstate Biotechnology (Lake Placid, NY). Negativity is determined by the absence of the band.
  • phosphorylation of ErbB2 (HER2) receptor is assessed by immunohistochemistry using a phospho-specific anti-HER2 antibody (clone PN2A; Thor et al., J. Clin. Oncol., 18(18):3230-3239 (2000 )).
  • KIRA ELISA U.S. Patent Nos. 5,766,863 ; 5,891,650 ; 5,914,237 ; 6,025,145 ; and 6,287,784
  • mass spectrometry comparing size of phosphorylated and non-phosphorylated HER2
  • e-tag proximity assay with anti-HER2 antibody e.g ., using the eTagTM assay kit available from Aclara BioSciences (Mountain View, CA). Details of the eTagTM assay are described hereinabove.
  • the ErbB2 antigen to be used for production of antibodies may be, e.g ., a soluble form of the extracellular domain of ErbB2 or a portion thereof, containing the desired epitope.
  • cells expressing ErbB2 at their cell surface e.g ., NIH-3T3 cells transformed to overexpress ErbB2; or a carcinoma cell line such as SK-BR-3 cells, see Stancovski et al., PNAS (USA), 88:8691-8695 (1991 )
  • Other forms of ErbB2 useful for generating antibodies will be apparent to those skilled in the art.
  • a protein that is immunogenic in the species to be immunized e.g ., keyhole limpet hemocyanin, serum albumin, bovine thy
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g ., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i . e ., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975 ), or may be made by recombinant DNA methods ( U.S. Patent No. 4,816,567 ).
  • a mouse or other appropriate host animal such as a hamster
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell ( Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986 )).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies ( Kozbor, J. Immunol., 133:3001 (1984 ); and Brön et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987 )).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980 ).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods ( Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986 )). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g ., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein.
  • Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262
  • monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990 ). Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., J. Mol. Biol., 222:581-597 (1991 ) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences ( U.S. Patent No. 4,816,567 ; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984 )), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers ( Jones et al., Nature, 321:522-525 (1986 ); Riechmann et al., Nature, 332:323-327 (1988 ); Verhoeyen et al., Science, 239:1534-1536 (1988 )), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies ( U.S.
  • Patent No. 4,816,567 wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody ( Sims et al., J. Immunol., 151:2296 (1993 ); Chothia et al., J. Mol. Biol., 196:901 (1987 )).
  • Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies ( Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992 ); Presta et al., J. Immunol., 151:2623 (1993 )).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i . e ., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • Exemplary humanized anti-ErbB2 antibodies which bind ErbB2 and block ligand activation of an ErbB receptor are described in WO 01/0245 , which is incorporated herein by reference.
  • the humanized antibodies of particular interest herein block EGF, TGF- ⁇ and/or HRG mediated activation of MAPK essentially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof) and/or binds ErbB2 essentially as effectively as murine monoclonal antibody 2C4 (or a Fab fragment thereof).
  • the humanized antibodies herein may, for example, comprise nonhuman hypervariable region residues incorporated into a human variable heavy domain and may further comprise a framework region (FR) substitution at a position selected from the group consisting of 69H, 71H and 73H utilizing the variable domain numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
  • the humanized antibody comprises FR substitutions at two or all of positions 69H, 71H and 73H.
  • An exemplary humanized antibody of interest herein comprises variable heavy domain complementarity determining residues GFTFTDYTMX, where X is preferably D or S (SEQ ID NO:7); DVNPNSGGSIYNQRFKG (SEQ ID NO:8); and/or NLGPSFYFDY (SEQ ID NO:9), optionally comprising amino acid modifications of those CDR residues, e.g ., where the modifications essentially maintain or improve affinity of the antibody.
  • the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above variable heavy CDR sequences.
  • Such antibody variants may be prepared by affinity maturation, e.g ., as described below.
  • the most preferred humanized antibody comprises the variable heavy domain amino acid sequence in SEQ ID NO:4 ( Figure 1B ).
  • the humanized antibody may comprise variable light domain complementarity determining residues KASQDVSIGVA (SEQ ID NO:10); SASYX1X2X3, where X1 is preferably R or L, X2 is preferably Y or E, and X3 is preferably T or S (SEQ ID NO:11); and/or QQYYIYPYT (SEQ ID NO:12), e.g ., in addition to those variable heavy domain CDR residues in the preceding paragraph.
  • Such humanized antibodies optionally comprise amino acid modifications of the above CDR residues, e.g ., where the modifications essentially maintain or improve affinity of the antibody.
  • the antibody variant of interest may have from about one to about seven or about five amino acid substitutions in the above variable light CDR sequences.
  • Such antibody variants may be prepared by affinity maturation, e.g ., as described below.
  • the most preferred humanized antibody comprises the variable light domain amino acid sequence in SEQ ID NO:3 ( Figure 1A ).
  • the present application also contemplates affinity matured antibodies which bind ErbB2 and block ligand activation of an ErbB receptor.
  • the parent antibody may be a human antibody or a humanized antibody, e.g ., one comprising the variable light and/or heavy sequences of SEQ ID Nos. 3 and 4, respectively ( i.e ., variant 574; Figure 1A and B ).
  • the affinity matured antibody preferably binds to ErbB2 receptor with an affinity superior to that of murine 2C4 or variant 574 (e.g ., from about two or about four fold, to about 100 fold or about 1000 fold improved affinity, e.g ., as assessed using a ErbB2-extracellular domain (ECD) ELISA).
  • ECD ErbB2-extracellular domain
  • variable heavy CDR residues for substitution include H28, H30, H34, H35, H64, H96, H99, or combinations of two or more ( e.g ., two, three, four, five, six, or seven of these residues).
  • variable light CDR residues for alteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 or combinations of two or more ( e.g ., two to three, four, five or up to about ten of these residues).
  • the humanized antibody or affinity matured antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate.
  • the humanized antibody or affinity matured antibody may be an intact antibody, such as an intact IgG1 antibody.
  • human antibodies can be generated.
  • transgenic animals e.g ., mice
  • transgenic animals e.g ., mice
  • JH antibody heavy-chain joining region
  • transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g ., Jakobovits et al., Proc. Natl. Acad. Sci.
  • phage display technology McCafferty et al., Nature, 348:552-553 (1990 )
  • V domain gene repertoires from unimmunized donors.
  • antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B-cell.
  • Phage display can be performed in a variety of formats; for their review see, e.g ., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology, 3:564-571 (1993 ).
  • V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991 ) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222:581-597 (1991 ), or Griffith et al., EMBO J., 12:725-734 (1993 ). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905 .
  • human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275 ).
  • these fragments were derived via proteolytic digestion of intact antibodies (see, e.g ., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992 ); and Brennan et al., Science, 229:81 (1985 )).
  • these fragments can now be produced directly by recombinant host cells.
  • the antibody fragments can be isolated from the antibody phage libraries discussed above.
  • Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments ( Carter et al., Bio/Technology, 10:163-167 (1992 )).
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185 ; U.S. Patent No. 5,571,894 ; and U.S. Patent No. 5,587,458 .
  • the antibody fragment may also be a ⁇ linear antibody ⁇ , e.g. , as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the ErbB2 protein. Other such antibodies may combine an ErbB2 binding site with binding site(s) for EGFR, ErbB3 and/or ErbB4.
  • an anti-ErbB2 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g ., CD2 or CD3), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the ErbB2-expressing cell.
  • a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g ., CD2 or CD3), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the ErbB2-expressing cell.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express ErbB2.
  • bispecific antibodies possess an ErbB2-binding arm and an arm which binds the cytotoxic agent (e.g ., saporin, anti-interferon- ⁇ vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten).
  • cytotoxic agent e.g ., saporin, anti-interferon- ⁇ vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g . F(ab')2 bispecific antibodies).
  • WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc ⁇ RIII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-Fc ⁇ RI antibody. A bispecific anti-ErbB2/Fc ⁇ antibody is shown in WO 98/02463 .
  • U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
  • bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities ( Millstein et al., Nature, 305:537-539 (1983 )). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 , and in Traunecker et al., EMBO J., 10:3655-3659 (1991 ).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690 . For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986 ).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g ., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones ( e.g ., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells ( U.S. Patent No. 4,676,980 ), and for treatment of HIV infection ( WO 91/00360 , WO 92/200373 , and EP 03089 ).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980 , along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229:81 (1985 ) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. J. Immunol., 147:60 (1991 ).
  • Amino acid sequence modification(s) of the anti-ErbB2 antibodies are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibodies.
  • Amino acid sequence variants of the anti-ErbB2 antibodies are prepared by introducing appropriate nucleotide changes into the anti-ErbB2 antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-ErbB2 antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processes of the anti-ErbB2 antibodies, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the anti-ErbB2 antibodies that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells, Science, 244:1081-1085 (1989 ).
  • a residue or group of target residues are identified (e.g ., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with ErbB2 antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-ErbB2 antibody variants are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an anti-ErbB2 antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of the anti-ErbB2 antibody molecules include the fusion to the N- or C-terminus of the anti-ErbB2 antibodies to a reporter molecule, an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the anti-ErbB2 antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • cysteine residues not involved in maintaining the proper conformation of the anti-ErbB2 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • a particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g ., a humanized or human antibody).
  • a parent antibody e.g ., a humanized or human antibody.
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g ., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle.
  • the phage-displayed variants are then screened for their biological activity (e.g ., binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variation alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • Nucleic acid molecules encoding amino acid sequence variants of the anti-ErbB2 antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-ErbB2 antibody.
  • ADCC antigen-dependent cell-mediated cyotoxicity
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J.
  • Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research, 53:2560-2565 (1993 ).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989 ).
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g ., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the ability of the antibody to block ErbB ligand binding to cells expressing the ErbB receptor may be determined. For example, cells naturally expressing, or transfected to express, ErbB receptors of the ErbB hetero-oligomer may be incubated with the antibody and then exposed to labeled ErbB ligand. The ability of the anti-ErbB2 antibody to block ligand binding to the ErbB receptor in the ErbB hetero-oligomer may then be evaluated.
  • inhibition of HRG binding to MCF7 breast tumor cell lines by anti-ErbB2 antibodies may be performed using monolayer MCF7 cultures on ice in a 24-well-plate format essentially as described in WO 01/00245 .
  • Anti-ErbB2 monoclonal antibodies may be added to each well and incubated for 30 minutes.
  • 125I-labeled rHRG ⁇ 1177-224 25 pm
  • Dose response curves may be prepared and an IC50 value may be calculated for the antibody of interest.
  • the antibody which blocks ligand activation of an ErbB receptor will have an IC50 for inhibiting HRG binding to MCF7 cells in this assay of about 50nM or less, more preferably 10nM or less.
  • the IC50 for inhibiting HRG binding to MCF7 cells in this assay may, for example, be about 100nM or less, more preferably 50mM or less.
  • the ability of the anti-ErbB2 antibody to block ErbB ligand-stimulated tyrosine phosphorylation of an ErbB receptor present in an ErbB hetero-oligomer may be assessed.
  • cells endogenously expressing the ErbB receptors or transfected to express them may be incubated with the antibody and then assayed for ErbB ligand-dependent tyrosine phosphorylation activity using an anti-phosphotyrosine monoclonal (which is optionally conjugated with a detectable label).
  • the kinase receptor activation assay described in U.S. Patent No. 5,766,863 is also available for determining ErbB receptor activation and blocking of that activity by an antibody.
  • one may screen for an antibody which inhibits HRG stimulation of p180 tyrosine phosphorylation in MCF7 cells essentially as described in Example 1 below.
  • the MCF7 cells may be plated in 24-well plates and monoclonal antibodies to ErbB2 may be added to each well and incubated for 30 minutes at room temperature; then rHRG ⁇ 1177-244 may be added to each well to a final concentration of 0.2 nM, and the incubation may be continued for 8 minutes. Media may be aspirated from each well, and reactions may be stopped by the addition of 100 ⁇ l SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl, pH 6.8).
  • Each sample (25 ⁇ l) may be electrophoresed on a 4-12% gradient gel (Novex) and then electrophoretically transferred to polyvinylidene difluoride membrane.
  • Antiphosphotyrosine (at 1 ⁇ g/ml) immunoblots may be developed, and the intensity of the predominant reactive band at Mr ⁇ 180,000 may be quantified by reflectance densitometry.
  • the antibody selected will preferably significantly inhibit HRG stimulation of p180 tyrosine phosphorylation to about 0-35% of control in this assay.
  • a dose-response curve for inhibition of HRG stimulation of p 180 tyrosine phosphorylation as determined by reflectance densitometry may be prepared and an IC50 for the antibody of interest may be calculated.
  • the antibody which blocks ligand activation of an ErbB receptor will have an IC50 for inhibiting HRG stimulation of p180 tyrosine phosphorylation in this assay of about 50nM or less, more preferably 10nM or less.
  • the IC50 for inhibiting HRG stimulation of p 180 tyrosine phosphorylation in this assay may, for example, be about 100nM or less, more preferably 50nM or less.
  • MDA-MB-175 cells may also assess the growth inhibitory effects of the antibody on MDA-MB-175 cells, e.g., essentially as described in Schaefer et al. Oncogene, 15:1385-1394 (1997 ).
  • MDA-MB-175 cells may treated with an anti-ErbB2 monoclonal antibody (10 ⁇ g/mL) for 4 days and stained with crystal violet.
  • Incubation with an anti-ErbB2 antibody may show a growth inhibitory effect on this cell line similar to that displayed by monoclonal antibody 2C4.
  • exogenous HRG will not significantly reverse this inhibition.
  • the antibody will be able to inhibit cell proliferation of MDA-MB-175 cells to a greater extent than monoclonal antibody 4D5 (and optionally to a greater extent than monoclonal antibody 7F3), both in the presence and absence of exogenous HRG.
  • the anti-ErbB2 antibody of interest may block heregulin dependent association of ErbB2 with ErbB3 in both MCF7 and SK-BR-3 cells as determined in a co-immunoprecipitation experiment such as that described in Example 1 substantially more effectively than monoclonal antibody 4D5, and preferably substantially more effectively than monoclonal antibody 7F3.
  • the growth inhibitory antibody of choice is able to inhibit growth of SK-BR-3 cells in cell culture by about 20-100% and preferably by about 50-100% at an antibody concentration of about 0.5 to 30 ⁇ g/ml.
  • the SK-BR-3 assay described in U.S. Patent No. 5,677,171 can be performed. According to this assay, SK-BR-3 cells are grown in a 1:1 mixture of F12 and DMEM medium supplemented with 10% fetal bovine serum, glutamine and penicillin streptomycin.
  • the SK-BR-3 cells are plated at 20,000 cells in a 35mm cell culture dish (2mls/35mm dish). 0.5 to 30 ⁇ g/ml of the anti-ErbB2 antibody is added per dish. After six days, the number of cells, compared to untreated cells are counted using an electronic COULTERTM cell counter. Those antibodies which inhibit growth of the SK-BR-3 cells by about 20-100% or about 50-100% may be selected as growth inhibitory antibodies.
  • the preferred assay is the PI uptake assay using BT474 cells.
  • BT474 cells which can be obtained from the American Type Culture Collection (Rockville, MD)
  • D-MEM Dulbecco's Modified Eagle Medium
  • Ham's F-12 50:50
  • heat-inactivated FBS Hyclone
  • 2 mM L-glutamine 2 mM L-glutamine
  • the BT474 cells are seeded at a density of 3 x 106 per dish in 100 x 20 mm dishes and allowed to attach overnight. The medium is then removed and replaced with fresh medium alone or medium containing 10 ⁇ g/ml of the appropriate monoclonal antibody. The cells are incubated for a 3 day time period. Following each treatment, monolayers are washed with PBS and detached by trypsinization.
  • Cells are then centrifuged at 1200 rpm for 5 minutes at 40C, the pellet resuspended in 3 ml ice cold Ca 2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ) and aliquoted into 35 mm strainer-capped 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 ⁇ g/ml). Samples may be analyzed using a FACSCANTM flow cytometer and FACSCONVERTTM CellQuest software (Becton Dickinson). Those antibodies which induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing antibodies.
  • an annexin binding assay using BT474 cells is available.
  • the BT474 cells are cultured and seeded in dishes as discussed in the preceding paragraph.
  • the medium is then removed and replaced with fresh medium alone or medium containing 10 ⁇ g/ml of the monoclonal antibody.
  • monolayers are washed with PBS and detached by trypsinization.
  • Cells are then centrifuged, resuspended in Ca 2+ binding buffer and aliquoted into tubes as discussed above for the cell death assay. Tubes then receive labeled annexin (e.g ., annexin V-FTIC) (1 ⁇ g/ml).
  • labeled annexin e.g ., annexin V-FTIC
  • Samples may be analyzed using a FACSCANTM flow cytometer and FACSCONVERTTM CellQuest software (Becton Dickinson). Those antibodies which induce statistically significant levels of annexin binding relative to control are selected as apoptosis-inducing antibodies.
  • a DNA staining assay using BT474 cells is available.
  • BT474 cells which have been treated with the antibody of interest as described in the preceding two paragraphs are incubated with 9 ⁇ g/ml HOECHST 33342TM for 2 hr at 370C, then analyzed on an EPICS ELITETM flow cytometer (Coulter Corporation) using MODFIT LTTM software (Verity Software House).
  • Antibodies which induce a change in the percentage of apoptotic cells which is 2 fold or greater (and preferably 3 fold or greater) than untreated cells (up to 100% apoptotic cells) may be selected as pro-apoptotic antibodies using this assay.
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioactive isotope (i.e ., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof), or a radioactive isotope (i.e ., a radioconjugate).
  • Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, a maytansine ( U.S. Patent No. 5,208,020 ), a trichothene, and CC1065 are also contemplated herein.
  • an antibody is conjugated to one or more maytansine molecules (e.g ., about 1 to about 10 maytansine molecules per antibody molecule).
  • Maytansine may, for example, be converted to May-SS-Me which may be reduced to May-SH3 and reacted with modified antibody ( Chari et al., Cancer Research, 52:127-131 (1992 )) to generate a maytansinoid-antibody immunoconjugate.
  • Another immunoconjugate of interest comprises an anti-ErbB2 antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ 1I, ⁇ 2I, ⁇ 3I, N-acetyl- ⁇ 1I, PSAG and ⁇ I1 ( Hinman et al., Cancer Research, 53:3336-3342 (1993 ) and Lode et al., Cancer Research, 58:2925-2928 (1998 )). See, also, U.S. Patent Nos. 5,714,586 ; 5,712,374 ; 5,264,586 ; and 5,773,001 expressly incorporated herein by reference.
  • Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993 .
  • the present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g ., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
  • a compound with nucleolytic activity e.g ., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
  • radioactive isotopes are available for the production of radioconjugated anti-ErbB2 antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu.
  • Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-di
  • a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238:1098 (1987 ).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/11026 .
  • the linker may be a ⁇ cleavable linker ⁇ facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker Chari et al., Cancer Research, 52:127-131 (1992 ) may be used.
  • fusion protein comprising an anti-ErbB2 antibody and cytotoxic agent may be made, e.g ., by recombinant techniques or peptide synthesis.
  • an antibody may be conjugated to a "receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g ., avidin) which is conjugated to a cytotoxic agent (e.g ., a radionucleotide).
  • a "ligand” e.g ., avidin
  • cytotoxic agent e.g ., a radionucleotide
  • ADPT Antibody Dependent Enzyme Mediated Prodrug Therapy
  • the antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g ., a peptidyl chemotherapeutic agent, see WO 81/01145 ) to an active anti-cancer drug.
  • a prodrug e.g ., a peptidyl chemotherapeutic agent, see WO 81/01145
  • a prodrug e.g ., a peptidyl chemotherapeutic agent, see WO 81/01145
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; ⁇
  • antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328:457-458 (1987 )).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
  • the enzymes of this invention can be covalently bound to the anti-ErbB2 antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g ., Neuberger et al., Nature, 312:604-608 (1984 ).
  • the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g ., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody may also or alternatively be linked to one or more of a variety of different moieties, such as a fluorescent label, a moiety with a known electrophoretic mobility, or a moiety that is able to cleave a specific linker molecule.
  • the antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the anti-ErbB2 antibodies disclosed herein may also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985 ); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980 ); U.S. Patent Nos. 4,485,045 and 4,544,545 ; and WO 97/38731 published October 23, 1997 . Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556 .
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982 ) via a disulfide interchange reaction.
  • a chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19):1484 (1989 ).
  • the invention also provides isolated nucleic acid encoding the antibodies, including humanized anti-ErbB2 antibodies, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g ., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the anti-ErbB2 antibodies may be produced recombinantly not only directly, but also as fusion polypeptides with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed ( i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.
  • yeast secretion the native signal sequence may be substituted by, e.g ., the yeast invertase leader, ⁇ factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646 .
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the anti-ErbB2 antibody.
  • Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Selection genes may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g ., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g ., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
  • suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-ErbB2 antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
  • host cells transformed or co-transformed with DNA sequences encoding anti-ErbB2 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g ., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199 .
  • a suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 ( Stinchcomb et al., Nature, 282:39 (1979 )).
  • the trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977 ).
  • the presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
  • vectors derived from the 1.6 ⁇ m circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts.
  • an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990 ).
  • Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991 ).
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the anti-ErbB2 antibody nucleic acid.
  • Promoters suitable for use with prokaryotic hosts include the phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
  • trp tryptophan
  • Other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the anti-ErbB2 antibody.
  • S.D. Shine-Dalgarno
  • Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Suitable vectors and promoters for use in yeast expression are further described in EP 73,657 .
  • Yeast enhancers also are advantageously used with yeast promoters.
  • Anti-ErbB2 antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g ., the action promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446 .
  • a modification of this system is described in U.S. Patent No. 4,601,978 .
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the anti-ErbB2 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-ErbB2 antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g ., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g ., Salmonella typhimurium, Serratia, e.g ., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g ., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g ., Salmonella typhimurium
  • Serratia
  • E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-ErbB2 antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • Kluyveromyces hosts such as, e.g ., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g ., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated anti-ErbB2 antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g ., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • vertebrate cells have been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977 )); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980 )); mouse sertoli cells (TM4, Mather, Biol.
  • COS-7 monkey kidney CV1 line transformed by SV40
  • human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977 )
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells ( Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982 )); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for anti-ErbB2 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the anti-ErbB2 antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology, 10:163-167 (1992 ) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains ( Lindmark et al., J. Immunol. Meth., 62:1-13 (1983 )).
  • Protein G is recommended for all mouse isotypes and for human ⁇ 3 ( Guss et al., EMBO J., 5:15671575 (1986 )).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABXTMresin J. T. Baker, Phillipsburg, NJ is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations ( e.g ., from about 0-0.25M salt).
  • Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980 )), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Zn-protein complexes Zn-protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • Preferred lyophilized anti-ErbB2 antibody formulations are described in WO 97/04801 , expressly incorporated herein by reference.
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, EGFR-targeted drug, anti-angiogenic agent, and/or cardioprotectant.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g ., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides ( U.S. Patent No.
  • copolymers ofL-glutamic acid and ⁇ ethyl-L-glutamate copolymers ofL-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3-hydroxybutyric acid poly-D-(-)-3-hydroxybutyric acid.
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • anti-ErbB2 antibodies may be used to treat various diseases or disorders.
  • exemplary conditions or disorders include benign or malignant tumors; leukemias and lymphoid malignancies; other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders.
  • anti-ErbB2 antibodies are used to treat tumors that are identified as responsive to treatment with such antibodies by the methods disclosed herein.
  • cancer examples include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g ., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as
  • the cancer to be treated is identified as responsive to treatment with anti-ErbB2 antibodies based on the identification of HER2/HER3 and/or HER2/HER1 heterodimers or the phosphorylation of ErbB receptor in a tumor sample.
  • a particular group of cancers where HER2/HER3 and/or HER2/HER1 heterodimer formation and/or phosphorylation of ErbB receptor is expected to be detected includes, without limitation lung breast cancer, lung cancer, ovarian cancer, including advanced, refractory or recurrent ovarian cancer, prostate cancer, colorectal cancer, and pancreatic cancer.
  • the cancer will generally comprise ErbB2-expressing cells, such that the anti-ErbB2 antibody herein is able to bind to the cancer. While the cancer may be characterized by overexpression of the ErbB2 receptor, the present application further provides a method for treating cancer which is not considered to be an ErbB2-overexpressing cancer.
  • various diagnostic/prognostic assays are available.
  • ErbB2 overexpression may be analyzed by IHC, e.g. using the HERCEPTEST ® (Dako). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a ErbB2 protein staining intensity criteria as follows:
  • FISH assays such as the INFORMTM (sold by Ventana, Arizona) or PATHVISIONTM (Vysis, Illinois) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of ErbB2 overexpression in the tumor.
  • the cancer will be one which expresses (and may, but does not have to, overexpress) EGFR.
  • cancers which may express/overexpress EGFR include squamous cell cancer (e.g ., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • squamous cell cancer e.g ., epi
  • the present invention is specifically suitable for the identification or breast cancer, prostate cancer, such as Castration-Resistant Prostate Cancer (CRPC), and ovarian cancer patients that are likely to respond well to treatment with an anti-HER2 antibody that blocks ligans activation of an ErbB heterodimer comprising HER2, such as monoclonal antibody 2C4 or rhuMAb 2C4.
  • CRPC Castration-Resistant Prostate Cancer
  • the cancer to be treated herein may be one characterized by excessive activation of an ErbB receptor, e.g. EGFR. Such excessive activation may be attributable to overexpression or increased production of the ErbB receptor or an ErbB ligand.
  • a diagnostic or prognostic assay will be performed to determine whether the patient's cancer is characterized by excessive activation of an ErbB receptor. For example, ErbB gene amplification and/or overexpression of an ErbB receptor in the cancer may be determined.
  • Assays for determining such amplification/overexpression are available in the art and include the IHC, FISH and shed antigen assays described above.
  • levels of an ErbB ligand, such as TGF- ⁇ in or associated with the tumor may be determined according to known procedures. Such assays may detect protein and/or nucleic acid encoding it in the sample to be tested. In one embodiment, ErbB ligand levels in the tumor may be determined using immunohistochemistry (IHC); see, for example, Scher et al., Clin. Cancer Research, 1:545-550 (1995 ). Alternatively, or additionally, one may evaluate levels of ErbB ligand-encoding nucleic acid in the sample to be tested; e.g ., via FISH, southern blotting, or PCR techniques.
  • IHC immunohistochemistry
  • ErbB receptor or ErbB ligand overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g ., by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g ., a radioactive isotope) and externally scanning the patient for localization of the label.
  • a detectable label e.g ., a radioactive isotope
  • the cancer to be treated is hormone independent cancer
  • expression of the hormone (e.g , androgen) and/or its cognate receptor in the tumor may be assessed using any of the various assays available, e.g ., as described above.
  • the patient may be diagnosed as having hormone independent cancer in that they no longer respond to anti-androgen therapy.
  • an immunoconjugate comprising the anti-ErbB2 antibody conjugated with a cytotoxic agent is administered to the patient.
  • the immunoconjugate and/or ErbB2 protein to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugates in killing the cancer cell to which it binds.
  • the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
  • the antibody administered is rhuMAb 2C4, or a functional equivalent thereof.
  • RhuMAb 2C4 is a humanized monoclonal antibody based on human IgG1 framework sequences and consisting of two heavy chains (449 residues) and two light chains (214 residues).
  • RhuMAb 2C4 differs significantly from another anti-HER2 antibody HERCEPTIN® (Trastuzumab) in the epitope-binding regions of the light chain and heavy chain.
  • HERCEPTIN® Trastuzumab
  • rhuMAb 2C4 binds to a completely different epitope on HER2.
  • the present invention provides sensitive methods for identifying cancers responsive to treatment with rhuMAb 2C4 or functional equivalents thereof. It is noted that such cancers responsive to rhuMAb 2C4 treatment are not required to overexpress HER2.
  • the anti-ErbB2 antibodies or immunoconjugates are administered to a human patient in accord with known methods, such as intravenous administration, e.g ., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred.
  • Other therapeutic regimens may be combined with the administration of the anti-ErbB2 antibody.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the patient is treated with two different anti-ErbB2 antibodies.
  • the patient may be treated with a first anti-ErbB2 antibody which blocks ligand activation of an ErbB receptor or an antibody having a biological characteristic of monoclonal antibody 2C4 as well as a second anti-ErbB2 antibody which is growth inhibitory (e.g. HERCEPTIN ® ) or an anti-ErbB2 antibody which induces apoptosis of an ErbB2-overexpressing cell ( e.g ., 7C2, 7F3 or humanized variants thereof).
  • growth inhibitory e.g. HERCEPTIN ®
  • an anti-ErbB2 antibody which induces apoptosis of an ErbB2-overexpressing cell (e.g ., 7C2, 7F3 or humanized variants thereof).
  • HERCEPTIN ® growth inhibitory
  • an anti-ErbB2 antibody which induces apoptosis of an ErbB2-overexpressing cell (e.g ., 7C
  • the patient may first be treated with rhuMAb 2C4 and then receive HERCEPTIN ® therapy.
  • the patient may be treated with both rhuMAb 2C4 and HERCEPTIN ® simultaneously.
  • the anti-ErbB2 antibody or antibodies may also be desirable to combine administration of the anti-ErbB2 antibody or antibodies, with administration of an antibody directed against another tumor associated antigen.
  • the other antibody in this case may, for example, bind to EGFR, ErbB3, ErbB4, or vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the treatment of the present invention involves the combined administration of an anti-ErbB2 antibody (or antibodies) and one or more chemotherapeutic agents or growth inhibitory agents, including coadministration of cocktails of different chemotherapeutic agents.
  • Preferred chemotherapeutic agents include taxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics.
  • Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992 ).
  • the antibody may be combined with an anti-hormonal compound; e.g ., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812 ); or an anti-androgen such as flutamide, in dosages known for such molecules.
  • an anti-hormonal compound e.g ., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812 ); or an anti-androgen such as flutamide
  • an anti-hormonal compound e.g ., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812 ); or an anti-androgen such as flutamide
  • the cancer to be treated is hormone independent cancer
  • the patient may previously have been subjected to anti-hormonal therapy and, after the cancer becomes hormone independent, the anti-Erb
  • a cardioprotectant to prevent or reduce myocardial dysfunction associated with the therapy
  • one or more cytokines may be beneficial to also coadminister to the patient.
  • a cardioprotectant to prevent or reduce myocardial dysfunction associated with the therapy
  • one or more cytokines may be beneficial to also coadminister to the patient.
  • the patient may be subjected to surgical removal of cancer cells and/or radiation therapy.
  • anti-ErbB2 antibodies herein may also be combined with an EGFR-targeted drug such as those discussed above in the definitions section resulting in a complementary, and potentially synergistic, therapeutic effect.
  • additional drugs which can be combined with the antibody include chemotherapeutic agents such as carboplatin, a taxane (e.g ., paclitaxel or docetaxel), gemcitabine, navelbine, cisplatin, oxaliplatin, or combinations of any of these such as carboplatin/docetaxel; another anti-HER2 antibody (e.g ., a growth inhibitory anti-HER2 antibody such as HERCEPTIN®, or an anti-HER2 antibody which induces apoptosis such as 7C2 or 7F3, including humanized or affinity matured variants thereof); a farnesyl transferase inhibitor; an anti-angiogenic agent (e.g ., an anti-VEGF antibody); an EGFR-targeted drug ( e.g ., C225 or ZD1839); a cytokine (e.g ., IL-2, IL-12, G-CSF or GM-CSF); or combinations of the above.
  • Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-ErbB2 antibody.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the preferred dosage of the antibody will be in the range from about 0.05mg/kg to about 10mg/kg.
  • one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g ., every week or every three weeks ( e.g ., such that the patient receives from about two to about twenty, e.g ., about six doses of the anti-ErbB2 antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-ErbB2 antibody.
  • an initial loading dose of about 4 mg/kg
  • a weekly maintenance dose of about 2 mg/kg of the anti-ErbB2 antibody.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • rhuMAb 2C4 is administered in a fixed dose of 420 mg (equivalent to doses of 6 mg/kg for a 70-kg subject) every 3 weeks. Treatment may start with a higher loading dose (e.g ., 840 mg, equivalent to 12 mg/kg of body weight) in order to achieve steady state serum concentrations more rapidly.
  • a higher loading dose e.g ., 840 mg, equivalent to 12 mg/kg of body weight
  • the present application contemplates administration of the antibody by gene therapy.
  • administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody”. See, for example, WO 96/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
  • nucleic acid (optionally contained in a vector) into the patient's cells
  • in vivo and ex vivo the nucleic acid is injected directly into the patient, usually at the site where the antibody is required.
  • ex vivo treatment the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g ., U.S. Patent Nos. 4,892,538 and 5,283,187 ).
  • U.S. Patent Nos. 4,892,538 and 5,283,187 There are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • a commonly used vector for ex vivo delivery of the gene is a retrovirus.
  • the currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example).
  • viral vectors such as adenovirus, Herpes simplex I virus, or adeno-associated virus
  • lipid-based systems useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g ., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem., 262:4429-4432 (1987 ); and Wagner et al., Proc. Natl. Acad. Sci. USA, 87:3410-3414 (1990 ).
  • an article of manufacture containing materials useful for the treatment of the disorders described above is provided.
  • the article of manufacture comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an anti-ErbB2 antibody.
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the label or package inserts indicates that the composition comprising the antibody which binds ErbB2 can be used to treat a patient suffering from a tumor in which the presence of HER2/HER1 and/or HER2/HER3 and/or HER2/HER4 complexes have been identified, and/or phosphorylation of ErbB receptor has been detected.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a first antibody which binds ErbB2 and inhibits growth of cancer cells which overexpress ErbB2; and (b) a second container with a composition contained therein, wherein the composition comprises a second antibody which binds ErbB2 and blocks ligand activation of an ErbB receptor.
  • the article of manufacture in this embodiment of the invention may further comprises a package insert indicating that the first and second antibody compositions can be used to treat cancer characterized by the presence of HER2/HER1 and/or HER2/HER3 and/or HER2/HER4 heterodimers, and/or by the phosphorylation of ErbB receptor.
  • the package insert may instruct the user of the composition (comprising an antibody which binds ErbB2 and blocks ligand activation of an ErbB receptor) to combine therapy with the antibody and any of the adjunct therapies described in the preceding section ( e.g .
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • the antibodies may also be used in diagnostic assays.
  • the antibodies are labeled as described in the methods above.
  • the antibodies of the present invention can be provided in a kit, i.e ., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g ., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g ., a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers (e.g ., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • hybridoma cell lines have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (ATCC): Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 October 17, 1996 7F3 ATCC HB-12216 October 17, 1996 4D5 ATCC CRL 10463 May 24, 1990 2C4 ATCC HB-12697 April 8, 1999
  • the murine monoclonal antibody 2C4 which specifically binds the extracellular domain of ErbB2 is described in WO 01/89566 , the disclosure of which is hereby expressly incorporated by reference in its entirety.
  • the cells were washed briefly with phosphate buffered saline (PBS) and then incubated with either 100 nM of the indicated antibody diluted in 0.2% w/v bovine serum albumin (BSA), RPMI medium, with 10 mM HEPES, pH 7.2 (binding buffer), or with binding buffer alone (control). After one hour at room temperature, HRG was added to a final concentration of 5 nM to half the wells (+). A similar volume of binding buffer was added to the other wells (-). The incubation was continued for approximately 10 minutes.
  • BSA bovine serum albumin
  • binding buffer pH 7.2
  • ErbB2 was immunoprecipitated using a monoclonal antibody covalently coupled to an affinity gel (Affi-Prep 10, Bio-Rad). This antibody (Ab-3, Oncogene Sciences, USA) recognizes a cytoplasmic domain epitope. Immunoprecipitation was performed by adding 10 ⁇ l of gel slurry containing approximately 8.5 ⁇ g of immobilized antibody to each lysate, and the samples were allowed to mix at room temperature for two hours. The gels were then collected by centrifugation. The gels were washed batchwise three times with lysis buffer to remove unbound material. SDS sample buffer was then added and the samples were heated briefly in a boiling water bath.
  • Preincubation with HERCEPTIN ® decreased the ErbB3 signal in MCF7 lysates but had little or no effect on the amount of ErbB3 co-precipitated from SK-BR-3 lysates.
  • Preincubation with an antibody against the EGF receptor had no effect on the ability of ErbB3 to co-immunoprecipitate with ErbB2 in either cell line.
  • tumor models Approximately 40 tumor models have been tested for responsiveness to 2C4. These models represent major cancers such as breast, lung, prostate and colon. 50-60% of the models responded to 2C4 treatment. Table 1 below lists selected tumor models tested for responsivity to 2C4. Briefly, human tumor xenograft fragments of about 3 mm size were transplanted underneath the skin of athymic nude mice. Alternatively, human tumor cells grown in vitro were detached from the culture dishes, resuspended in phosphate-buffered saline and subcutaneously injected into the flank of the immuno-compromised mice. The growth of tumors was monitored every 2 to 3 days using an electric caliper.
  • mice were randomized into different treatment and control groups. 2C4 was administered by intraperitoneal injection once every week. Control animals received equal volumes of vehicle solution containing no antibody on the same schedule as the treatment groups. The study was terminated after approximately 3 - 6 weeks, when the tumors of the control group reached a size of about 1000 - 1500 mm3. Responsiveness to treatment was defined as ⁇ 50% tumor volume reduction.
  • the models represent two major tumor types, namely non-small cell lung cancer (NSCLC; models #1-13) and mammary cancer (models #14-18).
  • NSCLC non-small cell lung cancer
  • models #1-13 models #1-13
  • mammary cancer models #14-18.
  • NSCLC non-small cell lung cancer
  • Nine of the NSCLC models (#1-9) and all breast cancer models were derived by serial in vivo passage of human tumor fragments in immunodeficient mice.
  • the remaining NSCLC models (#10-13) are cell-based models in which in vivo tumor growth is induced by implantation of in vitro propagated cells into immunocompromised mice.
  • 2C4 responsive and non-responsive tumors were subjected to immunoprecipitation with anti-ErbB2 antibodies to assay for the presence of ErbB2-ErbB3 and EGFR-ErbB2 heterodimers. Unless otherwise stated the methods were performed according to Maniatis T. et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, USA, Cold Spring Harbor Laboratory Press, 1982 .
  • Anti-HER2, anti-HER3 and anti-HER1 antibodies were selected that did not cross react.
  • HER1, HER2, HER3 and HER4 receptors were expressed in human embryonic kidney (HEK) 293 cells. The cells were lysed using a TritonTM X100 (1% weight per volume) containing HEPES buffer (pH 7.5). Approximately 20 ⁇ g of total cell protein from control cells and HER1, HER2, HER3 and HER4 expressing cells was separated on an SDS gel and transferred to a nitrocellulose membrane by a semi-dry blotting procedure. After blocking with gelatin, a variety of anti-HER1, anti-HER2 and anti-HER3 antibodies were tested for their cross reactivity against other ErbB receptors. Antibodies selected for use in the experiments described below did not show any significant cross reactivity.
  • Fresh tumor samples were crushed mechanically on ice and lysed in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 10 % (w/v) glycerol, 1 % (w/v) TritonTM X-100, 1 mM PMSF, 10 ⁇ g/ml aprotinin and 0.4 mM orthovanadate. Completely lysed tumors were centrifuged several times until the supernatants were completely clear.
  • Immunoprecipitation proceeded by combining cleared tumor lysates (5-7 mg protein per lysate), 5 ⁇ g anti ErbB2 antibody (ab-3, mouse monoclonal; Cat.# OP15, Oncogene Inc., USA) and 50 ⁇ l protein G-coupled agarose in 1.5 ml Eppendorf reaction tubes. Upon addition of a one to twofold volume of 50 mM HEPES buffer, pH 7.5 containing 0.1 % (w/v) TritonTM X-100 the tubes were rotated for 3-4 hours at 4 °C followed by centrifugation.
  • the pellets were washed two to three times with 500 ⁇ l of 50 mM HEPES buffer pH 7.5 containing 0.1 % (w/v) TritonTM X-100.
  • An equal volume of 2x Lämmli sample buffer was added to the washed immunoprecipitate and the samples were heated for 5 min at 95°C.
  • the samples were separated by SDS-PAGE and transferred to nitrocellulose.
  • the presence of EGFR-ErbB2 and ErbB2-ErbB3 heterodimers was assessed by probing the blots with antibodies against EGFR (rabbit polyclonal antibody against EGFR, Upstate Inc., USA; Cat.
  • Figure 3 shows the results of these experiments.
  • the presence of ErbB2-ErbB3 and/or EGFR-ErbB2 heterodimers can be seen.
  • a correlation was observed between 2C4 responsiveness, as shown in Table 1, and the presence of ErbB2-ErbB3 and/or EGFR-ErbB2 heterodimers.
  • HER2 phosphorylation was assessed by immunoprecipitation of HER2 and Western blot analysis. Positivity was determined by the presence of the phospho-HER2 band of the gel. Negativity was determined by the absence of the band. HER2 phosphorylation was confirmed by immunohistochemistry using a phospho-specific anti-HER2 antibody (clone PN2A, Thor et al., J. Clin. Oncol., 18:3230-9 (2000 ).
  • rhuMAb 2C4 The efficacy of rhuMAb 2C4 was assessed in nine established non-small cell lung carcinoma (NSCLC) xenografted tumor models (LXFE 211, LXFA 289, LXFA 297, LXFE 397, LXFA 526, LXFL 529, LXFA 629, LXFA 1041, LXFL 1071, Oncotest GmbH, Freiburg, Germany).
  • Human tumor xenografts are being considered as the most relevant models for anticancer drug development since the patient's tumors are growing as a solid tumor, develop a stroma, vasculature, a central necrosis and show dome differentiation.
  • the xenografted tumor models resemble very closely the original tumors in histology and chemosensitivity.
  • HER2 could be immunoprecipitated from tumor extracts from eight of nine NSCLC tumors. To determine the activation status of HER2, these blots were then probed with anti-phosphotyrosine (anti-PY) antibody. As shown in the lower panel of Figure 4 , the three responsive tumors (LXFA 297, LXFA 629 and 1072) displayed strong HER2 activation.
  • a patient is identified as having non-small cell lung cancer (NSCLC) that is responsive to treatment with rhuMAb 2C4 if a tumor from the patient is found to comprise HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 complexes.
  • NSCLC non-small cell lung cancer
  • a tumor sample is obtained by biopsy or during surgical resection of the tumor.
  • the sample is then analyzed for the presence of HER2/HER3 and/or HER2/HER1 and/or HER2/HER4 heterodimers.
  • Tumor cells or cell lysates are contacted with an eTagTM that specifically binds HER2.
  • the eTagTM comprises a detectable moiety and a first binding moiety that is specific for HER2.
  • the detectable moiety is linked to the first binding moiety with a cleavable linker. After allowing time for binding, excess eTagTM is removed.
  • the tumor cells or cell lysates are contacted with a second binding compound that specifically binds HER1 or HER3 or HER4. Unbound compound is removed by washing.
  • the second binding compound is then activated. If the first binding compound and the second binding compound are in close proximity, the activated second binding compound cleaves the cleavable linker in the eTagTM to produce a free detectable moiety.
  • the identification of free detectable moiety in the sample indicates that the tumor comprises HER2/HER1 or HER2/HER3 or HER2/HER4 heterodimers.
  • rhuMAb 2C4 is administered intravenously (IV) weekly or every three weeks at 2 or 4 mg/kg, respectively, until disease progression.
  • the antibody is supplied as a multi-dose liquid formulation (20mL fill at a concentration of 20mg/mL or higher concentration).
  • Primary endpoints for efficacy include response rate, and safety. Secondary efficacy endpoints include: overall survival, time to disease progression, quality of life, and/or duration of response.
  • a biological sample comprising cancer cells is obtained from candidates for the treatment, e.g ., by biopsy of tumor tissue, aspiration of tumor cells from ascitic fluid, or any other method known in clinical practice.
  • the biological sample is analyzed for HER2 phosphorylation, e.g ., by immunoprecipitation and Western blot analysis, and/or for the presence of HER2/HER3, HER2/HER1 and/or HER2/HER4 heterodimers by any of the techniques described above.
  • subjects diagnosed with ovarian cancer will undergo a biopsy of tumor tissue or aspiration of tumor cells from ascites fluid. This tissue will be analyzed for HER2 phosphorylation by immunoprecipitation and Western blot analysis. This will require a minimum of about 250 mg tumor tissue.
  • the patient Upon determination that the patient suffers from cancer (such as, Castration-Resistant Prostate Cancer - CRPC, or ovarian cancer) that is positive for HER2 phosphorylation, the patient will receive a loading dose of 840 mg of rhuMAb 2C4 on day 1 of cycle 1 (first 21-day treatment period), followed by 420 mg on day 1 of each subsequent 21-day cycle, as continuous intravenous infusion. Treatment continues by intravenous infusion every 3 weeks for up to one year (17 cycles), for patients who show no evidence of disease progression. Treatment can be discontinued any time earlier, for lack of response, adverse effects, or other reasons, at the discretion of the physician.
  • cancer such as, Castration-Resistant Prostate Cancer - CRPC, or ovarian cancer

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8372396B2 (en) 2004-10-20 2013-02-12 Genetech, Inc. Antibody formulations
US12233177B2 (en) 2019-09-16 2025-02-25 Amgen Inc. Method for external sterilization of drug delivery device

Families Citing this family (116)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA9811162B (en) * 1997-12-12 2000-06-07 Genentech Inc Treatment with anti-ERBB2 antibodies.
US7625859B1 (en) 2000-02-16 2009-12-01 Oregon Health & Science University HER-2 binding antagonists
US7393823B1 (en) 1999-01-20 2008-07-01 Oregon Health And Science University HER-2 binding antagonists
EP1282443B1 (fr) 2000-05-19 2009-09-02 Genentech, Inc. Analyse de detection genique permettant d'ameliorer la probabilite d'une reponse efficace a une therapie du cancer basee sur un antagoniste d'erbb
US20040229380A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB heterodimers as biomarkers
US20090111127A1 (en) * 2002-05-21 2009-04-30 Monogram Biosciences Inc. Surface Receptor Complexes as Biomarkers
US7402397B2 (en) 2002-05-21 2008-07-22 Monogram Biosciences, Inc. Detecting and profiling molecular complexes
US20040229294A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB surface receptor complexes as biomarkers
JP2006523314A (ja) * 2003-04-01 2006-10-12 モノグラム バイオサイエンシズ，インコーポレーテッド バイオマーカーとしてのＥｒｂＢ表面受容体複合体
US7402398B2 (en) * 2003-07-17 2008-07-22 Monogram Biosciences, Inc. Measuring receptor homodimerization
US20100291594A1 (en) * 2003-07-17 2010-11-18 Laboratory Corporation Of America Holdings ErbB Surface Receptor Complexes as Biomarkers
NZ545873A (en) * 2003-08-11 2009-05-31 Monogram Biosciences Inc Detecting and profiling molecular complexes
US7402399B2 (en) 2003-10-14 2008-07-22 Monogram Biosciences, Inc. Receptor tyrosine kinase signaling pathway analysis for diagnosis and therapy
US7329495B2 (en) 2004-06-09 2008-02-12 Board Of Regents, The University Of Texas System Mutations in KIT confer imatinib resistance in gastrointestinal stromal tumors
GT200500155A (es) * 2004-06-16 2006-05-15 Terapia del càncer resistente al platino
KR20180091967A (ko) 2004-07-22 2018-08-16 제넨테크, 인크. Ｈｅｒ２ 항체 조성물
CA2583230A1 (fr) * 2004-10-05 2006-04-20 Oregon Health And Science University Compositions et procedes de traitement de maladie
MX2007004079A (es) * 2004-10-06 2007-06-15 Wellstat Biologics Corp Detecci??n de niveles elevados de prote??na her-2/neu en celulas cancerosas circulantes y tratamiento.
WO2006045991A1 (fr) * 2004-10-25 2006-05-04 Astrazeneca Ab Methode permettant de determiner si une tumeur va reagir a un traitement chimiotherapeutique
BRPI0518086A (pt) * 2004-12-07 2008-10-28 Genentech Inc métodos para tratamento de cáncer, métodos de avaliação de fosforilação ou ativação de her e método de identificação
UA94899C2 (ru) * 2005-01-21 2011-06-25 Дженентек, Инк. Фиксированное дозирование антител к her
EP1850874B1 (fr) * 2005-02-23 2013-10-16 Genentech, Inc. Extension de la periode menant à la progression de la maladie ou à la survie chez les patients atteints du cancer de l'ovaire par le pertuzumab
US20060204505A1 (en) * 2005-03-08 2006-09-14 Sliwkowski Mark X Methods for identifying tumors responsive to treatment with HER dimerization inhibitors (HDIs)
JP2006316040A (ja) 2005-05-13 2006-11-24 Genentech Inc Ｈｅｒｃｅｐｔｉｎ（登録商標）補助療法
US20070009976A1 (en) * 2005-07-06 2007-01-11 Helmut Lenz Detection of a target antigen irrespective of the presence or absence of a corresponding therapeutic antibody
PE20070207A1 (es) 2005-07-22 2007-03-09 Genentech Inc Tratamiento combinado de los tumores que expresan el her
US7700299B2 (en) 2005-08-12 2010-04-20 Hoffmann-La Roche Inc. Method for predicting the response to a treatment
JP5025724B2 (ja) 2006-04-18 2012-09-12 ウェルスタット バイオロジクス コーポレイション 循環腫瘍性細胞からのタンパク質の検出
CA2646497A1 (fr) * 2006-04-18 2007-10-25 Wellstat Biologics Corporation Detection de cellules endotheliales en circulation
EP2036584B1 (fr) * 2006-06-27 2015-01-21 Asahi Kasei Kabushiki Kaisha Substrat pour traitement de fluide biologique
BRPI0717416A2 (pt) 2006-09-21 2013-11-12 Prometheus Lab Inc Método para realizar um imunoensaio complexo de alta produtividade, e, arranjo
CA2676049C (fr) 2007-03-01 2018-04-10 Symphogen A/S Compositions d'anticorps recombinants diriges contre le recepteur du facteur de croissance epidermique
EP2132573B1 (fr) 2007-03-02 2014-04-23 Genentech, Inc. Elément de prévision de la réponse à un inhibiteur de her
US20090258005A1 (en) * 2007-05-29 2009-10-15 Trubion Pharmaceuticals Inc. Therapeutic compositions and methods
US20090304590A1 (en) * 2007-05-29 2009-12-10 Wyeth Therapeutic compositions and methods
DK2171090T3 (da) 2007-06-08 2013-06-10 Genentech Inc Genekspressionsmarkører for tumorresistens over for HER2-inhibitorbehandling
US9551033B2 (en) 2007-06-08 2017-01-24 Genentech, Inc. Gene expression markers of tumor resistance to HER2 inhibitor treatment
RU2519647C2 (ru) * 2007-07-13 2014-06-20 Нестек С.А. Выбор лекарственных средств для терапии рака легких с помощью матриц на основе антител
CA2705263A1 (fr) * 2007-11-09 2009-05-14 Novartis Ag Therapie combinee renfermant un anticorps antagoniste anti-cd 40 et du cyclophosphamide, de la doxorubicine, de la vincristine et de la prednisone (chop) en vue du traitement de cellules malignes de type b
AU2008328781A1 (en) * 2007-11-27 2009-06-04 Ablynx N.V. Amino acid sequences directed against heterodimeric cytokines and/or their receptors and polypeptides comprising the same
US10416162B2 (en) 2007-12-20 2019-09-17 Monogram Biosciences, Inc. Her2 diagnostic methods
JP5690593B2 (ja) * 2007-12-26 2015-03-25 ヴァクシネックス， インコーポレイテッド 抗ｃ３５抗体併用療法および方法
US20140127200A1 (en) * 2008-01-03 2014-05-08 The Scripps Research Institute Multispecific Antibody Targeting and Multivalency Through Modular Recognition Domains
TWI472339B (zh) 2008-01-30 2015-02-11 Genentech Inc 包含結合至ｈｅｒ２結構域ii之抗體及其酸性變異體的組合物
EP2250498B1 (fr) 2008-02-25 2012-10-31 Prometheus Laboratories Inc. Sélection d' un médicament pour le traitement du cancer du sein à partir des matrices d' anticorps
JP5800454B2 (ja) * 2008-05-23 2015-10-28 株式会社ノエビア 皮膚外用剤、経口剤、抗酸化剤、抗老化剤、及び免疫賦活剤
BRPI0812682A2 (pt) 2008-06-16 2010-06-22 Genentech Inc tratamento de cáncer de mama metastático
US9086414B2 (en) 2008-07-08 2015-07-21 George Mason Research Foundation, Inc. Phosphorylated C-ErbB2 as a superior predictive theranostic marker for the diagnosis and treatment of cancer
MX367196B (es) * 2008-08-05 2019-08-08 Alm Holding Co Proceso para el reciclamiento frio en el sitio utilizando asfalto en espuma y aditivo de lubricacion.
EP2331577B1 (fr) 2008-08-29 2017-06-07 Symphogen A/S Compositions de l'anticorps de recombinaison anti-récepteur du facteur de croissance anti-épidermique egfr
ES2637411T3 (es) 2008-12-01 2017-10-13 Laboratory Corporation Of America Holdings Métodos y ensayos para medir p95 Y/O p95 en una muestra y anticuerpos específicos para p95
WO2010083463A1 (fr) * 2009-01-15 2010-07-22 Laboratory Corporation Of America Holdings Procédés de détermination d'une réponse de patient par la mesure de l'expression de her-2
CA2749846C (fr) 2009-01-15 2018-08-07 Laboratory Corporation Of America Holdings Procedes permettant de determiner la reponse d'un patient par mesure de her-3
ES2572728T3 (es) 2009-03-20 2016-06-02 F. Hoffmann-La Roche Ag Anticuerpos anti-HER biespecíficos
WO2010132723A1 (fr) * 2009-05-14 2010-11-18 Prometheus Laboratories Inc. Biomarqueurs permettant de déterminer la sensibilité de cellules cancéreuses du sein à un traitement ciblant le récepteur her2
CA2761280A1 (fr) 2009-05-29 2010-12-02 F. Hoffmann-La Roche Ag Modulateurs de la signalisation her2 chez des patients exprimant her2 souffrant d'un cancer de l'estomac
EP2454598B1 (fr) 2009-07-15 2017-03-22 DiaTech Holdings, Inc. Sélection de médicaments pour la thérapie d' un cancer gastrique au moyen de réseaux à base d' anticorps
RU2411915C1 (ru) * 2009-10-05 2011-02-20 Константин Петрович Гайденко Способ диагностики больных, пораженных опухолями головного мозга
CN102892779B (zh) 2010-02-18 2016-12-21 基因泰克公司 神经调节蛋白拮抗剂及其在治疗癌症中的用途
WO2011146568A1 (fr) 2010-05-19 2011-11-24 Genentech, Inc. Prédiction de réponses à un inhibiteur de her
WO2012009705A1 (fr) 2010-07-15 2012-01-19 Zyngenia, Inc. Complexes liant ang-2 et leurs utilisations
TW201302793A (zh) 2010-09-03 2013-01-16 Glaxo Group Ltd 新穎之抗原結合蛋白
US9155802B2 (en) * 2010-11-01 2015-10-13 Symphogen A/S Pan-HER antibody composition
WO2012069466A1 (fr) 2010-11-24 2012-05-31 Novartis Ag Molécules multi-spécifiques
JP5766296B2 (ja) 2010-12-23 2015-08-19 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ ポリペプチド−ポリヌクレオチド複合体、およびエフェクター成分の標的化された送達におけるその使用
US9719995B2 (en) 2011-02-03 2017-08-01 Pierian Holdings, Inc. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
EP2714738B1 (fr) 2011-05-24 2018-10-10 Zyngenia, Inc. Complexes plurispécifiques multivalents et monovalents, et leurs utilisations
MX2014001766A (es) 2011-08-17 2014-05-01 Genentech Inc Anticuerpos de neuregulina y sus usos.
JP6186575B2 (ja) 2011-09-02 2017-08-30 ダイアテック ホールディングス， インコーポレイテッドＤｉａＴｅｃｈ Ｈｏｌｄｉｎｇｓ， Ｉｎｃ． 治療有効性を判定するためのシグナル経路タンパク質のプロファイリング
US9273143B2 (en) 2011-09-30 2016-03-01 Regeneron Pharmaceuticals, Inc. Methods and compositions comprising a combination of an anti-ErbB3 antibody and an anti-EGFR antibody
HK1200468A1 (en) 2011-09-30 2015-08-07 Regeneron Pharmaceuticals, Inc. Anti-erbb3 antibodies and uses thereof
US9828635B2 (en) * 2011-10-06 2017-11-28 Aveo Pharmaceuticals, Inc. Predicting tumor response to anti-ERBB3 antibodies
ES3023516T3 (en) 2011-10-14 2025-06-02 Hoffmann La Roche Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
WO2013063229A1 (fr) 2011-10-25 2013-05-02 The Regents Of The University Of Michigan Traitement par un agent de ciblage de her2 dans des cancers non amplifiés par her2 ayant des cellules souches cancéreuses exprimant her2
MX350957B (es) 2011-11-23 2017-09-27 Medimmune Llc Moleculas de union especificas para her3 y usos de las mismas.
CN106987620A (zh) 2011-11-30 2017-07-28 霍夫曼-拉罗奇有限公司 癌症中的erbb3突变
WO2013083810A1 (fr) 2011-12-09 2013-06-13 F. Hoffmann-La Roche Ag Identification de non-répondeurs aux inhibiteurs de her2
JP2015514710A (ja) 2012-03-27 2015-05-21 ジェネンテック， インコーポレイテッド Ｈｅｒ３阻害剤に関する診断及び治療
HK1202431A1 (en) 2012-05-02 2015-10-02 Symphogen A/S Humanized pan-her antibody compositions
TWI641619B (zh) 2012-06-25 2018-11-21 美商再生元醫藥公司 抗－ｅｇｆｒ抗體及其用途
EP2719706A1 (fr) 2012-10-15 2014-04-16 Universität Zürich Ligands de HER2 bispécifiques pour la thérapie du cancer
AU2013331763B2 (en) 2012-10-15 2018-02-15 Universitat Zurich Bispecific HER2 ligands for cancer therapy
WO2014083178A1 (fr) 2012-11-30 2014-06-05 F. Hoffmann-La Roche Ag Identification de patients ayant besoin d'une cothérapie par un inhibiteur de pd-l1
CA2907181C (fr) 2013-03-15 2023-10-17 Viktor Roschke Complexes multispecifiques multivalents et monovalents et leurs utilisations
JP6402173B2 (ja) * 2013-04-05 2018-10-10 ラボラトリー コーポレイション オブ アメリカ ホールディングス Ｈｅｒ３の検出に基づく癌の診断、予後予測、および処置を容易にするためのシステムおよび方法
MY173295A (en) 2013-04-16 2020-01-14 Genentech Inc Pertuzumab variants and evaluation thereof
US11305012B2 (en) 2013-09-24 2022-04-19 Medimmune, Llc Binding molecules specific for HER3 and uses thereof
WO2015100459A2 (fr) 2013-12-27 2015-07-02 Merrimack Pharmaceuticals, Inc. Profils de biomarqueur pour prédire les résultats d'une thérapie cancéreuse utilisant des inhibiteurs d'erbb3 et/ou des chimiothérapies
US10745490B2 (en) 2014-04-11 2020-08-18 Celldex Therapeutics, Inc. Anti-ErbB antibodies and methods of use thereof
RU2725093C2 (ru) 2014-04-25 2020-06-29 Дженентек, Инк. Способы лечения раннего рака молочной железы трастузумабом-mcc-dm1 и пертузумабом
CN105985435B (zh) * 2015-01-30 2019-10-15 嘉和生物药业有限公司 全人源her2抗体的突变抗体及其编码基因和应用
JP6822980B2 (ja) 2015-05-30 2021-01-27 ジェネンテック， インコーポレイテッド Ｈｅｒ２陽性転移性乳癌の治療方法
US10184006B2 (en) 2015-06-04 2019-01-22 Merrimack Pharmaceuticals, Inc. Biomarkers for predicting outcomes of cancer therapy with ErbB3 inhibitors
WO2017087280A1 (fr) 2015-11-16 2017-05-26 Genentech, Inc. Procédés de traitement d'un cancer positif her2
US20210311024A1 (en) * 2016-02-19 2021-10-07 Konica Minolta, Inc. Non-clinical test method characterized by quantitative evaluation of experimental animal specimen
US20190151346A1 (en) 2016-05-10 2019-05-23 INSERM (Institute National de la Santé et de la Recherche Médicale) Combinations therapies for the treatment of cancer
CN107446045A (zh) 2016-07-22 2017-12-08 北京天广实生物技术股份有限公司 一种抗her2的抗体、其药物组合物及用途
US10377833B2 (en) 2016-07-22 2019-08-13 Beijing Mabworks Biotech Co., Ltd. Bispecific anti-HER2 antibody
JP2019532999A (ja) 2016-11-04 2019-11-14 ジェネンテック， インコーポレイテッド Ｈｅｒ２陽性乳がんの治療
MX2019007801A (es) 2016-12-28 2019-10-30 Genentech Inc Tratamiento de cáncer avanzado con expresión de her2.
ES2835376T3 (es) 2017-01-17 2021-06-22 Genentech Inc Formulaciones de anticuerpos frente a HER2 subcutáneas
CN114984206A (zh) 2017-03-02 2022-09-02 基因泰克公司 Her2阳性乳腺癌的辅助治疗
KR20190140952A (ko) 2017-04-24 2019-12-20 제넨테크, 인크. 막관통 및 인접막 도메인에서의 erbb2/her2 돌연변이
EP3807640A4 (fr) * 2018-06-14 2022-04-13 Memorial Sloan Kettering Cancer Center Méthodes de prédiction de la réactivité de patients atteints d'un cancer du poumon à des thérapies ciblant her2
AU2019309913B2 (en) 2018-07-23 2025-02-27 Trevi Therapeutics, Inc. Treatment of chronic cough, breathlessness and dyspnea
SMT202500141T1 (it) 2018-09-06 2025-05-12 Daiichi Sankyo Co Ltd Coniugati farmaco-anticorpo di derivati dinucleotidici ciclici
JP2022504472A (ja) 2018-10-08 2022-01-13 ウニベルシテート チューリッヒ Ｈｅｒ２結合四量体ポリペプチド
WO2020132210A1 (fr) 2018-12-19 2020-06-25 Incyte Corporation Inhibiteurs de la voie jak1 pour le traitement d'une maladie gastro-intestinale
AU2020247828A1 (en) * 2019-03-22 2021-11-18 Certis Therapeutics Pty Ltd Anti-HER2 binding molecules
EP4087569A4 (fr) 2020-01-10 2024-01-24 Trevi Therapeutics, Inc. Méthodes d'administration de nalbuphine
WO2021188578A1 (fr) * 2020-03-16 2021-09-23 University Of Southern California Procédés de criblage permettant d'identifier des composés à petites molécules qui favorisent ou inhibent la croissance de cellules tumorales circulantes, et leurs utilisations
KR20230030621A (ko) 2020-06-29 2023-03-06 제넨테크, 인크. 퍼투주맙 + 트라스투주맙 고정 용량 조합
TW202241454A (zh) 2021-02-01 2022-11-01 日商第一三共股份有限公司 抗體－免疫賦活化劑共軛物之新穎製造方法
IL305816A (en) 2021-03-12 2023-11-01 Daiichi Sankyo Co Ltd Glycan and a method for preparing a medicine containing it
IL315341A (en) 2022-03-02 2024-10-01 Daiichi Sankyo Co Ltd METHOD FOR PRODUCING Fc-CONTAINING MOLECULE
CN119855836A (zh) 2022-09-09 2025-04-18 第一三共株式会社 新型寡糖、该寡糖的制造中间体、以及它们的制造方法

Citations (110)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
EP0003089A1 (fr) 1978-01-06 1979-07-25 Bernard David Séchoir pour feuilles imprimées par sérigraphie
WO1981001145A1 (fr) 1979-10-18 1981-04-30 Univ Illinois Medicaments "pro-drugs" pouvant etre actives par des enzymes hydrolytiques
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
WO1983000502A1 (fr) 1981-08-10 1983-02-17 Armour Dial Inc Procede de fabrication de savon
EP0073657A1 (fr) 1981-08-31 1983-03-09 Genentech, Inc. Préparation d'antigène de surface de l'hépatite B dans une levure
US4419446A (en) 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
EP0183070A2 (fr) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation de levures du genre Pichia
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
WO1987000195A1 (fr) 1985-06-28 1987-01-15 Celltech Limited Culture de cellules animales
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
EP0244234A2 (fr) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
WO1988007378A1 (fr) 1987-03-09 1988-10-06 Cancer Research Campaign Technology Ltd. Amelioration apportees a des systemes d'administration de medicaments
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (de) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Verfahren zur biotechnischen Herstellung van alkalischer Phosphatase
US4892538A (en) 1987-11-17 1990-01-09 Brown University Research Foundation In vivo delivery of neurotransmitters by implanted, encapsulated cells
WO1990003430A1 (fr) 1988-09-23 1990-04-05 Cetus Corporation Milieu de culture de cellules pour l'amelioration de la croissance des cellules, de la longivite de la culture et de l'expression du produit
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
US4933294A (en) 1984-01-30 1990-06-12 Icrf Patents Limited Method of detecting truncated epidermal growth factor receptors
US4943533A (en) 1984-03-01 1990-07-24 The Regents Of The University Of California Hybrid cell lines that produce monoclonal antibodies to epidermal growth factor receptor
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
US4968603A (en) 1986-12-31 1990-11-06 The Regents Of The University Of California Determination of status in neoplastic disease
WO1990013646A1 (fr) 1989-04-28 1990-11-15 Transgene S.A. Application de nouveaux fragments d'adn en tant que sequence codant pour un peptide signal pour la secretion de proteines matures par des levures recombinantes, cassettes d'expression, levures transformees et procede de preparation de proteines correspondant
US4975278A (en) 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
EP0402226A1 (fr) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Vecteurs de transformation de la levure yarrowia
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1991000360A1 (fr) 1989-06-29 1991-01-10 Medarex, Inc. Reactifs bispecifiques pour le traitement du sida
WO1991005264A1 (fr) 1989-09-29 1991-04-18 Oncogenetics Partners Detection et quantification des proteines apparentees neu dans les liquides biologiques des humains
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
WO1992020373A1 (fr) 1991-05-14 1992-11-26 Repligen Corporation Anticorps d'heteroconjugues pour le traitement des infections a l'hiv
US5183884A (en) 1989-12-01 1993-02-02 United States Of America Dna segment encoding a gene for a receptor related to the epidermal growth factor receptor
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
US5212290A (en) 1989-09-08 1993-05-18 The Johns Hopkins University Antibodies specific for type II mutant EGTR
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
WO1993021232A1 (fr) 1992-04-10 1993-10-28 Research Development Foundation IMMUNOTOXINES DIRIGEES CONTRE DES ANTIGENES DE SURFACE APPARENTEES A c-erbB-2(HER-2/neu)
WO1993021319A1 (fr) 1992-04-08 1993-10-28 Cetus Oncology Corporation ANTICORPS HUMANISES SPECIFIES PAR C-erbB-2
US5264586A (en) 1991-07-17 1993-11-23 The Scripps Research Institute Analogs of calicheamicin gamma1I, method of making and using the same
WO1993025673A1 (fr) 1992-06-04 1993-12-23 The Regents Of The University Of California Therapie genique in vivo a l'aide d'une sequence significative sans intron
WO1994000136A1 (fr) 1992-06-30 1994-01-06 Oncologix, Inc. COMBINAISON D'ANTICORPS MONOCLONAUX ANTI-erbB-2 ET PROCEDE POUR SON UTILISATION
US5283187A (en) 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
WO1994004690A1 (fr) 1992-08-17 1994-03-03 Genentech, Inc. Immunoadhesines bispecifiques
WO1994011026A2 (fr) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b
EP0599274A1 (fr) 1992-11-24 1994-06-01 Bristol-Myers Squibb Company HER4, récepteur tyrosine kinase de la famille du récepteur de facteur de croissance épidermique
EP0616812A1 (fr) 1993-03-24 1994-09-28 Berlex Biosciences Combinaison d'agents anti-hormonause et molécules de liaison pour le traitement du cancer
WO1994022478A1 (fr) 1993-03-30 1994-10-13 The Trustees Of The University Of Pennsylvania PREVENTION DE TUMEURS AVEC DES ANTICORPS MONOCLONAUX DIRIGES CONTRE L'ONCOGENE $i(NEU)
US5401638A (en) 1986-06-04 1995-03-28 Oncogene Science, Inc. Detection and quantification of neu related proteins in the biological fluids of humans
EP0659439A2 (fr) 1993-12-24 1995-06-28 MERCK PATENT GmbH Immunoconjugués
WO1995025167A1 (fr) 1994-03-17 1995-09-21 Merck Patent Gmbh Fragments d'anticorps a simple chaine anti-egfr et anticorps anti-egfr
WO1996003397A1 (fr) 1994-07-21 1996-02-08 Akzo Nobel N.V. Formulations de peroxides cetoniques cycliques
WO1996007321A1 (fr) 1994-09-06 1996-03-14 The Uab Research Foundation Procedes de modulation de la fonction proteine dans les cellules par utilisation d'homologues d'anticorps intracellulaires
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
WO1996016673A1 (fr) 1994-12-02 1996-06-06 Chiron Corporation Methode permettant d'induire une reponse immunitaire avec un anticorps bispecifique
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
WO1996033978A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
WO1996040210A1 (fr) 1995-06-07 1996-12-19 Imclone Systems Incorporated Anticorps et fragments d'anticorps inhibant la croissance des tumeurs
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
WO1997000271A1 (fr) 1995-06-14 1997-01-03 The Regents Of The University Of California Nouveaux anticorps humains a forte affinite diriges contre des antigenes tumoraux
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
WO1997004801A1 (fr) 1995-07-27 1997-02-13 Genentech, Inc. Formulation de proteine lyophilisee isotonique et stable
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5641869A (en) 1991-05-24 1997-06-24 Genentech, Inc. Method for purifying heregulin
WO1997035885A1 (fr) 1996-03-27 1997-10-02 Genentech, Inc. ANTICORPS DE LA PROTEINE ErbB3
US5677171A (en) 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
WO1997038731A1 (fr) 1996-04-18 1997-10-23 The Regents Of The University Of California Immunoliposomes optimisant l'internalisation dans des cellules cibles
WO1998002463A1 (fr) 1996-07-11 1998-01-22 Medarex, Inc. COMPOSES THERAPEUTIQUES A SPECIFICITE MULTIPLE CONSISTANT EN ANTICORPS ANTI-RECEPTEURS DU FC$g(a)
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5766863A (en) 1993-11-23 1998-06-16 Genentech, Inc. Kinase receptor activation assay
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
US5783186A (en) 1995-12-05 1998-07-21 Amgen Inc. Antibody-induced apoptosis
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
WO1998043960A1 (fr) 1997-04-03 1998-10-08 American Cyanamid Company 3-cyano quinolines substituees
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1998045479A1 (fr) 1997-04-04 1998-10-15 Albany Medical College Methode d'evaluation d'un cancer de la prostate
US5824311A (en) 1987-11-30 1998-10-20 Trustees Of The University Of Pennsylvania Treatment of tumors with monoclonal antibodies against oncogene antigens
WO1998050433A2 (fr) 1997-05-05 1998-11-12 Abgenix, Inc. Anticorps monoclonaux humains contre le recepteur du facteur de croissance epidermique
US5837234A (en) 1995-06-07 1998-11-17 Cytotherapeutics, Inc. Bioartificial organ containing cells encapsulated in a permselective polyether suflfone membrane
WO1999006378A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs irreversibles de tyrosines kinases
WO1999006396A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs bicycliques irreversibles de tyrosine kinases
WO1999009016A1 (fr) 1997-08-01 1999-02-25 American Cyanamid Company Derives de quinazoline substitues et leur utilisation en tant qu'inhibiteurs de la tyrosine kinase
US5891650A (en) 1993-11-23 1999-04-06 Genentech Incorporated Kinase receptor activation assay
US5891996A (en) 1972-09-17 1999-04-06 Centro De Inmunologia Molecular Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (EGF-R); diagnostic and therapeutic use
WO1999019488A1 (fr) 1997-10-15 1999-04-22 Children's Medical Center Corporation Nouveaux recepteurs humains de l'egf et leur utilisation
WO2001000245A2 (fr) 1999-06-25 2001-01-04 Genentech, Inc. Anticorps anti-erbb2 humanises et traitement a l'aide de ces anticorps
US6287784B1 (en) 1993-11-23 2001-09-11 Genentech, Inc. Kinase receptor activation assay
WO2001089566A1 (fr) 2000-05-19 2001-11-29 Genentech, Inc. Analyse de detection genique permettant d'ameliorer la probabilite d'une reponse efficace a une therapie du cancer basee sur un antagoniste d'erbb
US20010049105A1 (en) 1999-04-30 2001-12-06 Sharat Singh Methods employing generalized target-binding e-tag probes
WO2002018444A2 (fr) 2000-09-01 2002-03-07 Genentech, Inc. Antagonistes de erbb4
US20020064805A1 (en) * 1996-03-27 2002-05-30 Genentech, Inc. Isolated nucleic acids, vectors and host cells encoding ErbB3 antibodies
US6399743B1 (en) 1999-05-14 2002-06-04 Dept. Of Veterans Affairs Isolation and characterization of a rat epidermal growth factor related protein
US20030096373A1 (en) 1999-05-14 2003-05-22 Majumdar Adhip P. N. Antibodies to a novel EGF-receptor related protein (ERRP)

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US190689A (en) * 1877-05-15 Improvement in car-brakes
US126818A (en) * 1872-05-14 Improvement in carpet-stretchers
US229380A (en) * 1880-06-29 John charles
US197835A (en) * 1877-12-04 Improvement in dovetailing and grooving machines
US229294A (en) * 1880-06-29 woolnoug-h
US229293A (en) * 1880-06-29 Swinging can
US5811098A (en) * 1992-11-24 1998-09-22 Bristol-Myers Squibb Company Antibodies to HER4, human receptor tyrosine kinase
ZA988461B (en) * 1997-09-18 1999-03-30 Idec Pharma Corp Synergistic composition and methods for treating neoplastic or cancerous growths and for restoring or boosting hematopoiesis
KR100399475B1 (ko) * 1998-02-12 2003-09-29 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 순환 중인 암세포의 신속하고 효과적인 분리 방법 및 이를위한 제제
US6417168B1 (en) * 1998-03-04 2002-07-09 The Trustees Of The University Of Pennsylvania Compositions and methods of treating tumors
US6514700B1 (en) 1999-04-30 2003-02-04 Aclara Biosciences, Inc. Nucleic acid detection using degradation of a tagged sequence
US6627400B1 (en) * 1999-04-30 2003-09-30 Aclara Biosciences, Inc. Multiplexed measurement of membrane protein populations
PT2283866E (pt) * 1999-06-25 2015-07-20 Genentech Inc Métodos de tratamento utilizando conjugados de anticorpo anti-erbb-maitansinóide
US7041292B1 (en) * 1999-06-25 2006-05-09 Genentech, Inc. Treating prostate cancer with anti-ErbB2 antibodies
US6949245B1 (en) * 1999-06-25 2005-09-27 Genentech, Inc. Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies
KR100816958B1 (ko) * 1999-07-30 2008-03-26 메다렉스, 인코포레이티드 항-Ｆｃ 수용체 결합제를 포함하는 치료 화합물
SI1210115T1 (sl) * 1999-08-27 2009-12-31 Genentech Inc Doziranja za zdravljenje s protitelesi proti ErbB2
JP2004002211A (ja) * 2001-04-27 2004-01-08 Takeda Chem Ind Ltd 癌の予防・治療方法
US20040138160A1 (en) * 2001-04-27 2004-07-15 Kenichiro Naito Preventive/therapeutic method for cancer
US20030190689A1 (en) * 2002-04-05 2003-10-09 Cell Signaling Technology,Inc. Molecular profiling of disease and therapeutic response using phospho-specific antibodies
US20040229293A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui Surface receptor complexes as biomarkers
US20040229294A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB surface receptor complexes as biomarkers
US20040229380A1 (en) * 2002-05-21 2004-11-18 Po-Ying Chan-Hui ErbB heterodimers as biomarkers
AU2003251597A1 (en) * 2002-06-19 2004-01-06 Abgenix, Inc. Method for predicting response to epidermal growth factor receptor-directed therapy
KR20050025669A (ko) * 2002-07-25 2005-03-14 아클라라 바이오사이언시스 인코퍼레이티드 수용체 올리고머화의 검출
UA94899C2 (ru) * 2005-01-21 2011-06-25 Дженентек, Инк. Фиксированное дозирование антител к her

Patent Citations (114)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US5891996A (en) 1972-09-17 1999-04-06 Centro De Inmunologia Molecular Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (EGF-R); diagnostic and therapeutic use
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
EP0003089A1 (fr) 1978-01-06 1979-07-25 Bernard David Séchoir pour feuilles imprimées par sérigraphie
WO1981001145A1 (fr) 1979-10-18 1981-04-30 Univ Illinois Medicaments "pro-drugs" pouvant etre actives par des enzymes hydrolytiques
US4419446A (en) 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
WO1983000502A1 (fr) 1981-08-10 1983-02-17 Armour Dial Inc Procede de fabrication de savon
EP0073657A1 (fr) 1981-08-31 1983-03-09 Genentech, Inc. Préparation d'antigène de surface de l'hépatite B dans une levure
US4601978A (en) 1982-11-24 1986-07-22 The Regents Of The University Of California Mammalian metallothionein promoter system
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (de) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Verfahren zur biotechnischen Herstellung van alkalischer Phosphatase
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4933294A (en) 1984-01-30 1990-06-12 Icrf Patents Limited Method of detecting truncated epidermal growth factor receptors
US4943533A (en) 1984-03-01 1990-07-24 The Regents Of The University Of California Hybrid cell lines that produce monoclonal antibodies to epidermal growth factor receptor
US4965199A (en) 1984-04-20 1990-10-23 Genentech, Inc. Preparation of functional human factor VIII in mammalian cells using methotrexate based selection
EP0183070A2 (fr) 1984-10-30 1986-06-04 Phillips Petroleum Company Transformation de levures du genre Pichia
WO1987000195A1 (fr) 1985-06-28 1987-01-15 Celltech Limited Culture de cellules animales
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
EP0244234A2 (fr) 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
US5401638A (en) 1986-06-04 1995-03-28 Oncogene Science, Inc. Detection and quantification of neu related proteins in the biological fluids of humans
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US4968603A (en) 1986-12-31 1990-11-06 The Regents Of The University Of California Determination of status in neoplastic disease
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
WO1988007378A1 (fr) 1987-03-09 1988-10-06 Cancer Research Campaign Technology Ltd. Amelioration apportees a des systemes d'administration de medicaments
US4892538A (en) 1987-11-17 1990-01-09 Brown University Research Foundation In vivo delivery of neurotransmitters by implanted, encapsulated cells
US5283187A (en) 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
US5824311A (en) 1987-11-30 1998-10-20 Trustees Of The University Of Pennsylvania Treatment of tumors with monoclonal antibodies against oncogene antigens
US5677171A (en) 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5772997A (en) 1988-01-12 1998-06-30 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US4975278A (en) 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
WO1990003430A1 (fr) 1988-09-23 1990-04-05 Cetus Corporation Milieu de culture de cellules pour l'amelioration de la croissance des cellules, de la longivite de la culture et de l'expression du produit
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
WO1990013646A1 (fr) 1989-04-28 1990-11-15 Transgene S.A. Application de nouveaux fragments d'adn en tant que sequence codant pour un peptide signal pour la secretion de proteines matures par des levures recombinantes, cassettes d'expression, levures transformees et procede de preparation de proteines correspondant
EP0402226A1 (fr) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Vecteurs de transformation de la levure yarrowia
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
WO1991000360A1 (fr) 1989-06-29 1991-01-10 Medarex, Inc. Reactifs bispecifiques pour le traitement du sida
US5212290A (en) 1989-09-08 1993-05-18 The Johns Hopkins University Antibodies specific for type II mutant EGTR
WO1991005264A1 (fr) 1989-09-29 1991-04-18 Oncogenetics Partners Detection et quantification des proteines apparentees neu dans les liquides biologiques des humains
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US5183884A (en) 1989-12-01 1993-02-02 United States Of America Dna segment encoding a gene for a receptor related to the epidermal growth factor receptor
US5480968A (en) 1989-12-01 1996-01-02 The United States Of America As Represented By The Department Of Health And Human Services Isolated polypeptide erbB-3, related to the epidermal growth factor receptor and antibody thereto
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
WO1992020373A1 (fr) 1991-05-14 1992-11-26 Repligen Corporation Anticorps d'heteroconjugues pour le traitement des infections a l'hiv
US5641869A (en) 1991-05-24 1997-06-24 Genentech, Inc. Method for purifying heregulin
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5264586A (en) 1991-07-17 1993-11-23 The Scripps Research Institute Analogs of calicheamicin gamma1I, method of making and using the same
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (fr) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions induisant la destruction de cellules infectees par l'hiv
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
WO1993016185A2 (fr) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Proteine de liaison biosynthetique pour marqueur de cancer
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
WO1993021319A1 (fr) 1992-04-08 1993-10-28 Cetus Oncology Corporation ANTICORPS HUMANISES SPECIFIES PAR C-erbB-2
WO1993021232A1 (fr) 1992-04-10 1993-10-28 Research Development Foundation IMMUNOTOXINES DIRIGEES CONTRE DES ANTIGENES DE SURFACE APPARENTEES A c-erbB-2(HER-2/neu)
WO1993025673A1 (fr) 1992-06-04 1993-12-23 The Regents Of The University Of California Therapie genique in vivo a l'aide d'une sequence significative sans intron
WO1994000136A1 (fr) 1992-06-30 1994-01-06 Oncologix, Inc. COMBINAISON D'ANTICORPS MONOCLONAUX ANTI-erbB-2 ET PROCEDE POUR SON UTILISATION
WO1994004690A1 (fr) 1992-08-17 1994-03-03 Genentech, Inc. Immunoadhesines bispecifiques
WO1994011026A2 (fr) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Application therapeutique d'anticorps chimeriques et radio-marques contre l'antigene a differentiation restreinte des lymphocytes b humains pour le traitement du lymphome des cellules b
EP0599274A1 (fr) 1992-11-24 1994-06-01 Bristol-Myers Squibb Company HER4, récepteur tyrosine kinase de la famille du récepteur de facteur de croissance épidermique
EP0616812A1 (fr) 1993-03-24 1994-09-28 Berlex Biosciences Combinaison d'agents anti-hormonause et molécules de liaison pour le traitement du cancer
WO1994022478A1 (fr) 1993-03-30 1994-10-13 The Trustees Of The University Of Pennsylvania PREVENTION DE TUMEURS AVEC DES ANTICORPS MONOCLONAUX DIRIGES CONTRE L'ONCOGENE $i(NEU)
US6287784B1 (en) 1993-11-23 2001-09-11 Genentech, Inc. Kinase receptor activation assay
US5891650A (en) 1993-11-23 1999-04-06 Genentech Incorporated Kinase receptor activation assay
US5766863A (en) 1993-11-23 1998-06-16 Genentech, Inc. Kinase receptor activation assay
US6025145A (en) 1993-11-23 2000-02-15 Genentech, Inc. Kinase receptor activation assay
US5914237A (en) 1993-11-23 1999-06-22 Genentech Incorporated Kinase receptor activation assay
EP0659439A2 (fr) 1993-12-24 1995-06-28 MERCK PATENT GmbH Immunoconjugués
WO1995025167A1 (fr) 1994-03-17 1995-09-21 Merck Patent Gmbh Fragments d'anticorps a simple chaine anti-egfr et anticorps anti-egfr
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
WO1996003397A1 (fr) 1994-07-21 1996-02-08 Akzo Nobel N.V. Formulations de peroxides cetoniques cycliques
WO1996007321A1 (fr) 1994-09-06 1996-03-14 The Uab Research Foundation Procedes de modulation de la fonction proteine dans les cellules par utilisation d'homologues d'anticorps intracellulaires
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
WO1996016673A1 (fr) 1994-12-02 1996-06-06 Chiron Corporation Methode permettant d'induire une reponse immunitaire avec un anticorps bispecifique
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
WO1996030347A1 (fr) 1995-03-30 1996-10-03 Pfizer Inc. Derives de quinazoline
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO1996033980A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
WO1996033978A1 (fr) 1995-04-27 1996-10-31 Zeneca Limited Derives de quinazoline
US5837234A (en) 1995-06-07 1998-11-17 Cytotherapeutics, Inc. Bioartificial organ containing cells encapsulated in a permselective polyether suflfone membrane
WO1996040210A1 (fr) 1995-06-07 1996-12-19 Imclone Systems Incorporated Anticorps et fragments d'anticorps inhibant la croissance des tumeurs
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
WO1997000271A1 (fr) 1995-06-14 1997-01-03 The Regents Of The University Of California Nouveaux anticorps humains a forte affinite diriges contre des antigenes tumoraux
WO1997004801A1 (fr) 1995-07-27 1997-02-13 Genentech, Inc. Formulation de proteine lyophilisee isotonique et stable
US5783186A (en) 1995-12-05 1998-07-21 Amgen Inc. Antibody-induced apoptosis
WO1997035885A1 (fr) 1996-03-27 1997-10-02 Genentech, Inc. ANTICORPS DE LA PROTEINE ErbB3
US20020064805A1 (en) * 1996-03-27 2002-05-30 Genentech, Inc. Isolated nucleic acids, vectors and host cells encoding ErbB3 antibodies
WO1997038983A1 (fr) 1996-04-12 1997-10-23 Warner-Lambert Company Inhibiteurs irreversibles de tyrosine kinases
WO1997038731A1 (fr) 1996-04-18 1997-10-23 The Regents Of The University Of California Immunoliposomes optimisant l'internalisation dans des cellules cibles
WO1998002463A1 (fr) 1996-07-11 1998-01-22 Medarex, Inc. COMPOSES THERAPEUTIQUES A SPECIFICITE MULTIPLE CONSISTANT EN ANTICORPS ANTI-RECEPTEURS DU FC$g(a)
WO1998043960A1 (fr) 1997-04-03 1998-10-08 American Cyanamid Company 3-cyano quinolines substituees
WO1998045479A1 (fr) 1997-04-04 1998-10-15 Albany Medical College Methode d'evaluation d'un cancer de la prostate
WO1998050433A2 (fr) 1997-05-05 1998-11-12 Abgenix, Inc. Anticorps monoclonaux humains contre le recepteur du facteur de croissance epidermique
WO1999006396A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs bicycliques irreversibles de tyrosine kinases
WO1999006378A1 (fr) 1997-07-29 1999-02-11 Warner-Lambert Company Inhibiteurs irreversibles de tyrosines kinases
WO1999009016A1 (fr) 1997-08-01 1999-02-25 American Cyanamid Company Derives de quinazoline substitues et leur utilisation en tant qu'inhibiteurs de la tyrosine kinase
WO1999019488A1 (fr) 1997-10-15 1999-04-22 Children's Medical Center Corporation Nouveaux recepteurs humains de l'egf et leur utilisation
US20010049105A1 (en) 1999-04-30 2001-12-06 Sharat Singh Methods employing generalized target-binding e-tag probes
US6399743B1 (en) 1999-05-14 2002-06-04 Dept. Of Veterans Affairs Isolation and characterization of a rat epidermal growth factor related protein
US20030096373A1 (en) 1999-05-14 2003-05-22 Majumdar Adhip P. N. Antibodies to a novel EGF-receptor related protein (ERRP)
WO2001000245A2 (fr) 1999-06-25 2001-01-04 Genentech, Inc. Anticorps anti-erbb2 humanises et traitement a l'aide de ces anticorps
WO2001089566A1 (fr) 2000-05-19 2001-11-29 Genentech, Inc. Analyse de detection genique permettant d'ameliorer la probabilite d'une reponse efficace a une therapie du cancer basee sur un antagoniste d'erbb
WO2002018444A2 (fr) 2000-09-01 2002-03-07 Genentech, Inc. Antagonistes de erbb4

Non-Patent Citations (230)

* Cited by examiner, † Cited by third party
Title
"A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"Antibodies, A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"Drug-Induced Heart Disease. New York", 1980, ELSEVIER, pages: 191 - 215
"Remington's Pharmaceutical Sciences", 1980
AASLAND ET AL., BR. J. CANCER, vol. 57, 1988, pages 358 - 363
AGUS D B ET AL: "Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth", SIGNALING NETWORKS AND CELL CYCLE CONTROL: THE MOLECULAR BASIS OF CANCER AND OTHER DISEASES, XX, XX, vol. 2, 1 August 2002 (2002-08-01), pages 127 - 137, XP002988666 *
ANDERSON ET AL., SCIENCE, vol. 256, 1992, pages 808 - 813
ARTEAGA ET AL., CANCER RES., vol. 54, 1994, pages 3758 - 3765
AZZOLI, C.G. ET AL., SEMIN. ONCOL., vol. 29, no. 4, 2002, pages 59 - 65
BACUS ET AL., CANCER RESEARCH, vol. 52, 1992, pages 2580 - 2589
BACUS ET AL., MOLECULAR CARCINOGENESIS, vol. 3, 1990, pages 350 - 362
BARNES ET AL., ANAL BIOCHEM., vol. 102, 1980, pages 25 5
BASELGA ET AL., J. CLIN. ONCOL., vol. 14, 1996, pages 737 - 744
BASELGA; MENDELSOHN, PHARMAC. THER., vol. 64, 1994, pages 127 - 154
BERGER ET AL.: "Immunodeficient Mice in Oncology", 1992, article "Establishment and Characterization of Human Tumor Xenografts in Thymus-Aplastic Nude Mice", pages: 23 - 46
BERGER, D. P.; WINTERHALTER, B. R.; FIEBIG, H. H.: "In Immunodeficient Mice in Oncology", 1992, article "Establishment and Characterization of Human Tumor Xenografts in Thymus-Aplastic Nude Mice", pages: 23 - 46
BORST ET AL., GYNECOL. ONCOL., vol. 38, 1990, pages 364
BRENNAN ET AL., SCIENCE, vol. 229, 1985, pages 81
BRENNAN, P.J. ET AL., ONCOGENE, vol. 19, 2000, pages 6093 - 6101
BROCKHOFF ET AL., CYTOMETRY, vol. 44, 2001, pages 338 - 48
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC, pages: 51 - 63
BRUGGENNANN ET AL., YEAR IN IMMUNO., vol. 7, 1993, pages 33
BURGER, A. M.; HARTUNG, G.; STEHLE, G.; SINN, H.; FIEBIG, H. H.: "Pre-clinical evaluation of a methotrexate-albumin conjugate (MTX-HSA) in human tumor xenografts in vivo", INT. J. CANCER, vol. 92, 2001, pages 718 - 24
CAPEL ET AL., IMMUNOMETHODS, vol. 4, 1994, pages 25 - 34
CARON ET AL., J. EXP MED., vol. 176, 1992, pages 1191 - 1195
CARPENTER ET AL., ANN. REV. BIOCHEM., vol. 56, 1987, pages 881 - 914
CARRAWAY ET AL., NATURE, vol. 387, 1997, pages 512 - 516
CARRAWAY; CANTLEY, CELL, vol. 78, 1994, pages 5 - 8
CARTER ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 163 - 167
CARTER ET AL., BIOLTECHNOLOGY, vol. 10, 1992, pages 163 - 167
CARTER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285
CHANG ET AL., NATURE, vol. 387, 1997, pages 509 - 512
CHARI ET AL., CANCER RESEARCH, vol. 52, 1992, pages 127 - 131
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901
CHOTHIA; LESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOW ET AL., CLIN. CANCER RES., vol. 7, 2001, pages 1957 - 1962
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
CLYNES ET AL., PNAS (USA), vol. 95, 1998, pages 652 - 656
COHEN ET AL., ONCOGENE, vol. 4, 1989, pages 81 - 88
COOK ET AL., MOL. CELL. BIOL., vol. 11, 1991, pages 2547 - 2557
CUNNINGHAM; WELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DE HAAS ET AL., J. LAB. CLIN. MED., vol. 126, 1995, pages 330 - 41
DIGIOVANNA ET AL., CANCER RES., vol. 62, 2002, pages 6667 - 6673
DREBIN ET AL., CELL, vol. 41, 1985, pages 695 - 706
DREBIN ET AL., NATURE, vol. 312, 1984, pages 545 - 548
DREBIN ET AL., ONCOGENE, vol. 2, 1988, pages 273 - 277
D'SOUZA ET AL., PROC. NATL. ACAD. SCI., vol. 91, 1994, pages 7202 - 7206
EARP ET AL., BREAST CANCER RESEARCH AND TREATMENT, vol. 35, 1995, pages 115 - 132
EPSTEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 3688
ESTEVA ET AL., PATHOL. ONCOL. RES., vol. 7, 2001, pages 171 - 177
FALLS ET AL., CELL, vol. 72, 1993, pages 801 - 815
FENDLY ET AL., CANCER RESEARCH, vol. 50, 1990, pages 1550 - 1558
FIEBIG ET AL.: "Contributions to Oncology: Relevance of Tumor Models for Anticancer Drug Development", vol. 54, 1999, article "Human Tumor Xenografts: Predictivity, Characterization and Discovery of New Anticancer Agents", pages: 29 - 50
FIEBIG, H. H.; BURGER, A. M.: "In Tumor Models in Cancer Research", 2002, HUMANA PRESS, article "Human Tumor Xenografts and Explants", pages: 113 - 137
FIEBIG, H. H.; DENGLER, W. A.; ROTH, T.: "In Contributions to Oncology: Relevance of Tumor Models for Anticancer Drug Development", 1999, article "Human Tumor Xenografts: Predictivity, Characterization and Discovery of New Anticancer Agents", pages: 29 - 50
FIEBIG; BURGER: "Models in Cancer Research", 2002, HUMANA PRESS, article "Human Tumor Xenografts and Explants", pages: 113 - 137
FLEER ET AL., BIOLTECHHOLOGY, vol. 9, 1991, pages 968 - 975
FUKUSHIGI ET AL., MOL CELL BIOL., vol. 6, 1986, pages 955 - 958
GABIZON ET AL., J. NATIONAL CANCER INST., vol. 81, no. 19, 1989, pages 1484
GADELLA; JOVIN, J. CELL BIOL., vol. 129, 1995, pages 1543 - 58
GAZZANO-SANTORO ET AL., J. IMMUNOL. METHODS, vol. 202, 1996, pages 163
GEURIN ET AL., ONCOGENE RES., vol. 3, 1988, pages 21 - 31
GODING: "Monoclonal Antibodies: Principles and Practice", 1986, ACADEMIC PRESS, pages: 59 - 103
GRAHAM ET AL., J. GEN VIROL., vol. 36, 1977, pages 59
GRAUS-PORTA ET AL., EMBO J., vol. 16, 1995, pages 1647 - 55
GRAUS-PORTA ET AL., EMBO J., vol. 16, 1997, pages 1647 - 55
GREEN ET AL., CANCER RESEARCH, vol. 54, 1994, pages 738 - 741
GRIDLEY, D. S.; ANDRES, M. L.; GARNER, C.; MAO, X. W.; SLATER, J. M.: "Evaluation of TNF-alpha effects on radiation efficacy in a human lung adenocarcinoma model", ONCOL RES., vol. 8, 1996, pages 485 - 95
GRIFFITH ET AL., EMBO J., vol. 12, 1993, pages 725 - 734
GROENEN ET AL., GROWTH FACTORS, vol. 11, 1994, pages 235 - 257
GRUBER ET AL., J. IMMUNOL., vol. 152, 1994, pages 5368
GU ET AL., CANCER LETT., vol. 99, 1996, pages 185 - 9
GUS ET AL., EMBO J, vol. 5, 1986, pages 15671575
GUYER ET AL., J. IMMUNOL., vol. 117, 1976, pages 587
HAM ET AL., METH. ENZ., vol. 58, 1979, pages 44
HANCOCK ET AL., CANCER RES., vol. 51, 1991, pages 4575 - 4580
HARARI ET AL., ONCOGENE, vol. 18, 1999, pages 2681 - 89
HARARI ET AL., ONCOGENE, vol. L8, 1999, pages 2681 - 89
HARWERTH ET AL., J. BIOL. CHEM., vol. 267, 1992, pages 15160 - 15167
HIGASHIYAMA ET AL., SCIENCE, vol. 251, 1991, pages 936 - 939
HINMAN ET AL., CANCER RESEARCH, vol. 53, 1993, pages 3336 - 3342
HJALMARSON ET AL., DRUGS, vol. 47, no. 14, 1994, pages 31 - 9
HO ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 14523 - 14532
HOLLINGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HOLMES ET AL., SCIENCE, vol. 256, 1992, pages 1205 - 1210
HUDZIAK ET AL., MOL. CELL. BIOL., vol. 9, no. (3), 1989, pages 1165 - 1172
HUDZIAK ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 7159 - 7163
HUMPHREY ET AL., PNAS (USA), vol. 87, 1990, pages 4207 - 4211
HUNTER ET AL., BIOCHEM. J., vol. 320, pages 847 - 53
HWANG ET AL., PROC. NATL ACAD. SCI. USA, vol. 77, 1980, pages 4030
JAKOBOVITS ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 2551
JOHNSON, KEVIN S.; CHISWELL, DAVID J., CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 3, 1993, pages 564 - 571
JONES ET AL., FEBS LETTERS, vol. 447, 1999, pages 227 - 231
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JONES, GENETICS, vol. 85, 1977, pages 12
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
KANNAN ET AL., J. BIOL. CHEM., vol. 272, no. 6, 1997, pages 3330 - 3335
KASPRZYK ET AL., CANCER RESEARCH, vol. 52, 1992, pages 2771 - 2776
KENWORTHY, METHODS, vol. 24, 2001, pages 289 - 96
KERN ET AL., CANCER RES., vol. 50, 1990, pages 5184
KIM ET AL., J. IMMUNOL., vol. 24, 1994, pages 249
KIMURA ET AL., NATURE, vol. 348, 1990, pages 257 - 260
KING ET AL., SCIENCE, vol. 229, 1985, pages 974
KLAPPER ET AL., ONCOGENE, vol. 14, 1997, pages 2099 - 2109
KLAPPER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 4995 - 5000
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
KOMURASAKI ET AL., ONCOGENE, vol. 15, 1997, pages 2841 - 2848
KOSTELNY ET AL., J. 1MMUUOL., vol. 148, no. 5, 1992, pages 1547 - 1553
KOTTS ET AL., IN VITRO, vol. 26, no. 3, 1990, pages 59A
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
KRAUS ET AL., PNAS (USA), vol. 86, 1989, pages 9193 - 9197
KUMAR ET AL., MOL. CELL. BIOL., vol. 11, no. 2, 1991, pages 979 - 986
LEE ET AL., PHARM. REV., vol. 47, 1995, pages 51 - 85
LEMKE, G., MOLEC. & CELL. NEUROSCI., vol. 7, 1996, pages 247 - 262
LEVI ET AL., JOURNAL OF NEUROSCIENCE, vol. 15, 1995, pages 1329 - 1340
LEWIS ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 37, 1993, pages 255 - 263
LEWIS ET AL., CANCER RES., vol. 56, 1996, pages 1457 - 1465
LEWIS ET AL., CANCER RESEARCH, vol. 56, 1996, pages 1457 - 1465
LINDMARK ET AL., J. IMMUNOL. METH., vol. 62, 1983, pages 1 - 13
LODE ET AL., CANCER RESEARCH, vol. 58, 1998, pages 2925 - 2928
M. IN DAËRON, ANNU. REV. IMMUNOL., vol. 15, 1997, pages 203 - 234
M.C. PERRY: "Chemotherapy Service Ed.", 1992, WILLIAMS & WILKINS
MAIER ET AL., CANCER RES., vol. 51, 1991, pages 5361 - 5369
MANIATIS T. ET AL.: "Molecular Cloning: A Laboratory Manual", 1982, COLD SPRING HARBOR LABORATORY PRESS
MARCHIONNI ET AL., NATURE, vol. 362, 1993, pages 312 - 318
MARKS ET AL., BIOLTEEHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MARQUARDT ET AL., SCIENCE, vol. 223, 1984, pages 1079 - 1082
MARTIN ET AL., J. BIOL. CHEM., vol. 257, 1982, pages 286 - 288
MASSEY, NATURE, vol. 328, 1987, pages 457 - 458
MASUI ET AL., CANCER RESEARCH, vol. 44, 1984, pages 1002 - 1007
MATHER ET AL., ANNALS NY. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 553
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554
MCCANN ET AL., CANCER, vol. 65, 1990, pages 88 - 92
MCKENZIE ET AL., ONCOGENE, vol. 4, 1989, pages 543 - 548
MILLSTEIN ET AL., NATURE, vol. 305, 1983, pages 537 - 539
MOORE ET AL., CYTOTECHNOLOGY, vol. 17, 1995, pages 1 - 11
MORIMOTO ET AL., JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS, vol. 24, 1992, pages 107 - 117
MORRISON ET AL., PROC. NATL ACAD. SCI. USA, vol. 81, 1984, pages 6851
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
MORRISSEY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 1431 - 1435
MUNSON ET AL., ANAL. BIOCHEM., vol. 107, 1980, pages 220
MURAKAMI ET AL.: "Cell cycle regulation, oncogenes, and antineoplastic drugs", 1995, WB SAUNDERS, pages: 13
MURTHY, ARCH. BIOCHEM. BIOPHYS., vol. 252, 1987, pages 549 - 560
MYERS ET AL., METH. ENZYM., vol. 198, 1991, pages 277 - 290
NAGY ET AL., CYTOMETRY, vol. 32, 1998, pages 120 - 131
NAGY ET AL., CYTOMETRY, vol. 32, 1998, pages 120 - 31
NEUBERGER ET AL., NATURE, vol. 312, 1984, pages 604 - 608
PARACCHINI ET AL., ANTICANCER RES., vol. 13, 1993, pages 1607 - 1612
PARK ET AL., CANCER RES., vol. 49, 1989, pages 6605
PELES ET AL., CELL, vol. 69, 1992, pages 205 - 216
PIETRAS ET AL., ONCOGENE, vol. 9, 1994, pages 1829 - 1838
PLFCKTHUN: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315
PLOWMAN ET AL., NATURE, vol. 366, 1993, pages 473 - 475
PLOWMAN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 1746 - 1750
PLÜCKTHUN, IMMUNOL. REVS., vol. 130, 1992, pages 151 - 188
PRESTA ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623
PRESTA, CURR. OP. STRUCT. BIOL., vol. 2, 1992, pages 593 - 596
RAVETCH; KINET, ANNU. REV. IMMUNOL., vol. 9, 1991, pages 457 - 92
REYES ET AL., NATURE, vol. 297, 1982, pages 598 - 601
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 327
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
ROSS ET AL., CANCER, vol. 79, 1997, pages 2162 - 70
ROSS ET AL., HUM. PATHOL., vol. 28, 1997, pages 827 - 33
SADASIVAN ET AL., J. UROL., vol. 150, 1993, pages 126 - 31
SARUP ET AL., GROWTH REGULATION, vol. 1, 1991, pages 72 - 82
SASADA ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 190, 1993, pages 1173
SAVAGE ET AL., J. BIOL. CHEM., vol. 247, 1972, pages 7612 - 7621
SCHAEFER ET AL., ONCOGENE, vol. 15, 1997, pages 1385 - 1394
SCHECTER ET AL., NATURE, vol. 312, 1984, pages 513
SCHER ET AL., CLIN. CANCER RESEARCH, vol. 1, 1995, pages 545 - 550
SCOTT ET AL., J. BIOL. CHEM., vol. 266, 1991, pages 14300 - 5
SEIFERT ET AL., THE ANNALS OF PHARMACOTHERAPY, vol. 28, 1994, pages 1063 - 1072
SELVIN, NAT. STRUCT. BIOL., vol. 7, 2000, pages 730 - 34
SEMBA ET AL., PNAS (USA), vol. 82, 1985, pages 6497 - 6501
SHADDY ET AL., AM. HEARTJ., vol. 129, 1995, pages 197 - 9
SHALABY ET AL., J. EXP. MED., vol. 175, 1992, pages 217 - 225
SHAWVER ET AL., CANCER RES., vol. 54, 1994, pages 1367 - 1373
SHEPARD ET AL., J. CLIN. IMMUNOL., vol. 11, no. 3, 1991, pages 117 - 127
SHING ET AL., SCIENCE, vol. 259, 1993, pages 1604 - 1607
SHOPES, B., J. IMMUNOL., vol. 148, 1992, pages 2918 - 2922
SHOYAB ET AL., SCIENCE, vol. 243, 1989, pages 1074 - 1076
SIAS ET AL., J. IMMUNOL. METHODS, vol. 132, 1990, pages 73 - 80
SIMS ET AL., J. 1MMUUOL., vol. 151, 1993, pages 2296
SINGAL ET AL., J. MOL. CELL CARDIOL., vol. 27, 1995, pages 1055 - 1063
SKERRA ET AL., CURR. OPINION IN IMMUNOL., vol. 5, 1993, pages 256 - 262
SLAMON ET AL., SCIENCE, vol. 235, 1987, pages 177 - 182
SLAMON ET AL., SCIENCE, vol. 244, 1989, pages 707 - 712
SLIWKOWSKI ET AL., J. BIOL. CHEM., vol. 269, no. 20, 1994, pages 14661 - 14665
STANCOVSKI ET AL., PNAS (USA), vol. 88, 1991, pages 8691 - 8695
STANCOVSKI ET AL., PNAS (USA), vol. 88, 1991, pages 869I - 8695
STEIN, R.; GOVINDAN, S. V.; CHEN, S.; REED, L.; SPIEGELMAN, H.; GRIFFITHS, G. L.; HANSEN, H. J.; GOLDENBERG, D. M.: "Successful therapy of a human lung cancer xenograft using MAb RS7 labeled with residualizing radioiodine", CRIT. REV. ONCOL. HEMATOL., vol. 39, 2001, pages 173 - 80
STELLA ET AL.: "Prodrugs: A Chemical Approach to Targeted Drug Delivery", 1985, HUMANA PRESS, pages: 247 - 267
STEVENSON ET AL., ANTI-CANCER DRUG DESIGN, 1989, pages 219 - 230
STINCHCOMB ET AL., NATURE, vol. 282, 1979, pages 39
SURESH ET AL., METHODS IN ENZYMOLOGY, vol. 121, 1986, pages 210
TAGLIABUE ET AL., INT. J. CANCER, vol. 47, 1991, pages 933 - 937
THOR ET AL., J CLIN. ONCOL., vol. 18, no. 18, 2000, pages 3230 - 3239
THOR ET AL., J. CLIN. ONCOL., vol. 18, 2000, pages 3230 - 9
THOR ET AL., J. CLIN. ONCOLOGY, vol. 18, 2000, pages 3230 - 3239
TOYODA ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 7495 - 7500
TRAUNECKER ET AL., EMBO J., vol. 10, 1991, pages 3655 - 3659
TUTT ET AL., J. IMMUNOL., vol. 147, 1991, pages 60
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216
VAN DEN BERG, BIOLTECHNOLOGY, vol. 8, 1990, pages 1 3 5
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
VITETTA ET AL., CANCER RESEARCH, vol. 54, 1994, pages 5301 - 5309
VITETTA ET AL., SCIENCE, vol. 238, 1987, pages 1098
WAGNER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 3410 - 3414
WATERHOUSE ET AL., NUC. ACIDS. RES., vol. 21, 1993, pages 2265 - 2266
WEINER ET AL., CANCER RES., vol. 50, 1990, pages 421 - 425
WILLIAMS ET AL., PATHIOBIOLOGY, vol. 59, 1991, pages 46 - 52
WILMAN: "Prodrugs in Cancer Chemotherapy", BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 14, 1986, pages 375 - 382
WOLFF ET AL., CANCER RESEARCH, vol. 53, 1993, pages 2560 - 2565
WU ET AL., J. BIOL. CHENZ., vol. 262, 1987, pages 4429 - 4432
WU ET AL., J. CLIN. INVEST., vol. 95, 1995, pages 1897 - 1905
XU ET AL., INT. J. CANCER, vol. 53, 1993, pages 401 - 408
YAMAMOTO ET AL., NATURE, vol. 319, 1986, pages 230 - 234
YAMORI, T.; SATO, S.; CHIKAZAWA, H.; KADOTA, T.: "Anti-tumor efficacy of paclitaxel against human lung cancer xenografts", JPN. J. CANCER RES., vol. 88, 1997, pages 1205 - 10
YANIV, NATURE, vol. 297, 1982, pages 17 - 18
YOKOTA ET AL., LANCET, vol. 1, 1986, pages 765 - 767
YONEMURA ET AL., CANCER RES., vol. 51, 1991, pages 1034
ZHANG ET AL., PNAS (USA), vol. 94, no. 18, 1997, pages 9562 - 7
ZHANG ET AL., PROC. NATL. ACAD. SCI., vol. 94, 1997, pages 9562 - 9567
ZHAU ET AL., MOL. CARCINOG., vol. 1, 1990, pages 354 - 357
ZOU, Y.; WU, Q. P.; TANSEY, W.; CHOW, D.; HUNG, M. C.; CHAMSANGAVEJ, C.; WALLACE, S.; LI, C.: "Effectiveness of water soluble poly(L-glutamic acid)-camptothecin conjugate against resistant human lung cancer xenografted in nude mice", INT. J ONCOL., vol. 18, 2001, pages 331 - 6
ZUCK ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 11122 - 27

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